The polypeptide of thrombopoetin (srw) dna encoding a polypeptide tro (options), a method of obtaining a polypeptide (options), pharmaceutical composition, method of treatment (options), the antibody to the polypeptide tro

 

(57) Abstract:

The invention relates to genetic engineering. The polypeptide of thrombopoetin (SRW) has a biological activity of a specific stimulus or increasing the formation of platelets. Amino acid sequence is shown in the description. DNA encoding a polypeptide SRW with the amino acid sequence shown in the description. Polypeptide TRO get his expression in a suitable host and subsequent isolation. Farmcampsite for treatment-related platelet disorders includes an effective amount of the polypeptide TRO and an acceptable carrier. Farbkomposition administered to a subject in an effective amount for treating disorders associated with platelet, for example, thrombocytopenia. Conducting the immunization of an animal with the polypeptide TRO and produce antibodies to this polypeptide. The invention allows efficient treatment of disorders associated with platelets. 12 C. and 22 C.p. f-crystals, 34 ill., 19 table.

This application is a partial continuation of U. S. Patent Application Serial (unknown), filed January 31, 1995, pending, which is a partial continuation of U. S. Patent Application Serial 08/361811, filed December 22, 1994, pending, kotalawela partial continuation of U. S. Patent Application Serial 08/278083, filed July 20, 1994, pending, which is a partial continuation of U. S. Patent Application Serial 08/221020 filed April 1, 1994, revoked, which is a partial continuation of U. S. Patent Application Serial 08/212164 filed March 14, 1994, now abandoned.

The scope of the invention

This invention relates to a new protein having the activity of a specific stimulus or increasing the formation of platelets in vivo or enhance the proliferation and differentiation of progenitor cells megakaryocytes, the DNA sequence that encodes the protein, and how to obtain it.

Background of the invention

The megakaryocytes are large rich cytoplasm of multinucleated cells that produce platelets and can be found mainly in the bone marrow. The megakaryocytes originate from pluripotent hematopoietic (blood-forming) stem cells in the bone marrow. Primitive pluripotent stem cell is differentiated to some extent in precursor cells megakaryocytes, which give rise to several generations of megakaryocytes. Precursor cells megakaryocytes proliferate and differentiate into megakaryocytes. Sah non-nuclear cytoplasmic fragments, namely, the platelets in the circulation. On average, one Mature megakaryocyte formed 2-4 thousand platelets. Although the mechanism of clot formation in many respects unclear, believe that the megakaryocytes are usually localized on the protein surface of the endothelium of the sinus in the bone marrow and produce cytoplasmic processes extending into the sinusoids, where the fragmentation of platelets.

There are a lot of uncertainties regarding the mechanism of clot formation, although it is possible that the megakaryocytes are present locally in the dermal layer of the shell venous sinus bone marrow and that the cytoplasm passes through the dermal membrane, forming astronomically ledge on the inner wall of the venous sinus, resulting in the ejection of platelets.

It was suggested that there is a specific function for the formation and regulation of clot formation in the hemopoiesis of megakaryocytes. In healthy people and animals remains an effective amount of platelets, although it is known that the introduction of antibodies to platelets of healthy animals, for example, the number of platelets decreases sharply, then their number is temporarily Uwe is the train in the field of clinics known that reduction in the number of platelets (thrombocytopenia) or an increase in the number of platelets (thrombocytosis) can be seen even with normal amounts of red blood cells and leukocytes.

Sometimes the most important feature is the formation of platelet thrombus in hemostatic hemostatic) mechanism. If the hemostatic mechanism is not functioning properly due to the decrease in the number of platelets that form a trend towards the emergence of hemostasis.

It has been suggested the presence of specific regulatory mechanism megakaryocytopoiesis and thrombopoiesis. Platelets are maintained in effective amounts in healthy people and normal animals. However, it is known that antibodies to platelets normal animal the number of platelets decreases sharply within a short period of time, then starts to increase and temporarily exceeds the normal level, but in the end returns to a normal level. It is known that in the clinical field, the reduction in the number of platelets (thrombocytopenia) or an increase in their number (thrombocytosis) occur even with normal amounts of red blood cells and leukocytes. However, doctor, involved in the formation of platelets (e.g., similar to erythropoietin in the formation of red blood cells).

The most important function of platelets is the formation of blood clot in hemostatic hemostatic) mechanism. Bleeding occurs when the normal function of the haemostatic mechanism broken thrombocytopenia. In the field of radiotherapy and chemotherapy of cancer thrombocytopenia caused by bone marrow depression is a fatal complication, and to prevent the propensity to bleeding in such patients make the transfusion of platelets. Transfusion of platelets is also used after bone marrow transplantation or gipoplasticheskaya anemia.

Platelets for use in such transfusions are prepared thrombocytapheresis from the blood of healthy blood donors, however, such platelets for use in transfusions have a short shelf life and can be contaminated with bacterial infection. The transfusion of platelets is a possible danger of falling in patients with dangerous viruses such as human immunodeficiency virus (H IV) or the different hepatitis viruses, the risk of formation of antibodies specific for the antigen glavnoe host (GVHD), due to an admixture of lymphocytes in platelets used for transfusion.

Consequently, it would be helpful if patients with thrombocytopenia can boost the formation of platelets and at the same time, you will be able to reduce their dependence on transfusions of platelets. In addition, if the thrombocytopenia in cancer patients can be correct or prevent such processing can be performed more safely, the intensity of treatment can be increased and can be expected to further improve the anti-cancer actions.

For these reasons, conducted a number of intensive studies on the isolation and identification of specific regulatory factors involved in the regulation of formation of megakaryocytes and platelets. According to in vitro studies, the regulatory factors that control megakaryocytopoiesis, roughly divided into the following two factors (see Williams et al., J Cell Physiol., vol. 110, pp. 101-104, 1982). Colony-stimulating factor megakaryocytes (Honey-CSF) is a regulatory factor that stimulates the proliferation and differentiation of CFU-MK with the formation of colonies of megakaryocytes in semi-solid culture medium. Another regulatory factor, called factor is PE or etc., operates mainly on immature or Mature megakaryocytes, enhancing their differentiation and maturation. Honey-Pot found in combination with Honey-CSF activity in some cases. In addition, since the number of platelets increased with the introduction of serum or plasma collected from experimental animals with induced thrombocytopenia, other normal animals, an assumption was made that there is a humoral factor, called trombidium (SRW), can increase the formation of platelets in vivo.

In recent years, some cytokines, genes which were cloned and tested for their ability to stimulate megakaryocytopoiesis and trombozitopoez. Human IL-3 stimulated the formation of colonies of megakaryocytes man (Bruno et al., Exp. Hematol., vol.16, pp.371-377, 1988) and, at least in monkeys, increasing the number of platelets (Donahue et al., Science, vol. 241, p. 1820, 1988). However, because IL-3 is a factor acting on the proliferation and differentiation of all hematopoietic cells, it should be distinguished from specific regulatory factors governing megakaryocytes and the formation of platelets. Human IL-6 not found Honey-CSF activity, but acted immature ivo IL-6 induced the formation of platelets and increased maturation and caused a shift towards higher ploidy of megakaryocytes of the bone marrow in primates, but also caused side effects such as weight loss, induction of acute phase protein (Asano et al. , Blood, vol. 78, pp. 1467-1475, 1991). IL-11 person not found Honey-CSF activity, but showed Meg-Pot activity and increased formation of platelets in mice (It et al., Blood, vol. 81, pp. 901-908, 1993). In addition, LIF (inhibitor migration of lymphocytes) human significantly increased platelet counts in primates (Mowag et al., Blood, vol. 81, pp.3226-3233, 1993), but its action in vitro on megakaryocytes was weak (Burstein et al., J Cell. Physiol., vol. 153, pp. 305-312, 1992).

Although you can expect the clinical application of these cytokines as factors increasing platelets, their functions are not specific for a number of generations of megakaryocytes and they cause side effects. Therefore, it was necessary the presence of increasing the number of platelet factor that is specific to the system megakaryocyte-platelet and causes less side effects for clinical application.

Meg-CSF, Meg-Pot or TPO activity was detected in the serum, plasma or urine of patients with thrombocytopenia or animals or in the culture supernatant some of cultured human cell lines. However, it is currently unknown due to these cytokines.

Hoffman et al. found that the serum of patients with gipoplasticheskaya anemia and amegakaryocytic thrombocytopenic purpura contained Meg-CSF activity, which significantly increased the formation of colonies of human megakaryocytes (Hoffman et al., N. Eng. J. Med., vol. 305, pp. 533-538, 1981). After that, Mazur et al. reported that Honey-CSF activity present in the serum of patients with aplastic anemia and how it differs from IL-3 and DM-CSF (azur et al., Blood, vol.76, pp.290-297, 1990). Similar Honey-CSF activity was found in the sera of cancer patients receiving intensive cytotoxic chemotherapy, and patients with bone marrow transplantation (Mazur et al. , Exp. Hematol., vol.12, pp.624-628, 1984; de Alarcon and Schmieder, Prog. Clin. Bio. Res., vol.215, pp.335-340, 1986).

Hoffman et al. reported that Meg-CSF with an average mol. weight of 46000 was purified from plasma of patients with hypoechogenicity thrombocytopenia (Hoffman et al. J. Clin. Invest.,vol 75, pp. 1174-1182, 1985), but further research showed that this material was not of such a level of purity that would allow precise amino acid sequencing (Hoffman, Blood, vol. 74, pp. 1196-1212, 1989). A substance with a TRO-like activity, which was strengthened by the inclusion of75Se-Selenomethionine in the newly formed platelets in mice, was partially purified from the plasma of thrombocytopeni Average mol. the mass obtained from plasma factor was 40000 (Grossi et al., Hematologica, vol. 72, pp. 291-295, 1987; Vannucchi et al., Leukemia, vol.2, pp. 236-240, 1988).

Meg-CSF and TPO-like activity were also detected in the urine of patients with aplastic anemia and severe ITP (Kawakita et al., Br. J. Haematol. vol. 48, pp. 609-615, 1981; Kawakita et al., Blood, vol. 556-560, 1983). Kawakita et al. also reported that the Meg-CSF activity detected in the extract of the urine of patients with aplastic anemia, found the average mol. the weight of 45,000, a gel filtration under dissociating conditions (Kawakita et al. , Br. J. Haematol., vol.62, pp.715-722, 1986). Erikson-Miller et al. also reported cleanup Meg-CSF of these urine samples, however, gave no information about its structure (Erikson-Miller et al., "Blood Cell Growth Factors: their present and future use in hematology and oncology" ed. by Murphy, AlphaMed Press, Dayton, Ohio, pp.204-220, 1992). Turner et al. purified factor stimulation of megakaryocytes (MSF), with Meg-CSF activity from the urine of patients with transplanted bone marrow and cloned its gene (Turner et al., Blood, vol.78, p.1106, 279a, 1991, (abstr., Suppl.1)). This MSF is saying. a lot 28000-35000. The identity of this factor with Meg-CSF, discovered so far in samples of serum and plasma thrombocytopenic patients, and its activity in increasing the number of platelets has yet to be clarified.

A substance with a TRO-like activity (mol. MCI) and its biological and biochemical properties were investigated intensively, however, its structure is still unknown (McDonald et al., J. Lab. Clin. Hed. vol. 106, pp. 162-174, 1985; McDonald, Int. J. Cell Cloning, vol. 7, pp. 139-155, 1989). Other researchers, in contrast, reported that the main activity in the conditioned medium of SOME cells, which enhances the maturation of megakaryocytes in vitro due to the known cytokines, namely, IL-6 and EPO (withy et al., J. Cell. Physiol., vol. 15, pp. 362-372, 1992).

With regard to obtained from animal factors, Evatt et al. reported that TPO-like activity, reinforcing the inclusion of75Se-Selenomethionine in the newly formed platelets in rabbits and mice, was detected in plasma thromocytopenia rabbits induced by injection of serum against platelets (Evatt et al., J. Lab. Clin. Med., vol. 83, pp. 364-371, 1974). In addition, a number of such studies have been published since the 60's through the 70-ies (e.g., dell et al., Proc. Soc. Biol. Med., vol. 108, pp. 428-431, 1961; Evatt and Levin, J. Clin. Invest., vol. 48, pp. 1615-1626; Harker, Am. J. Physiol., vol. 218, pp. 1376-1380, 1970; Shreiner and Levin. J. Clin Invest, vol.49, pp.1709-1713, 1970; and Penington, Br. Med. J. vol. 1, pp.606-608, 1970). Evatt et al., and Hill and Levin had a partial purification of the solid-like activity of plasma thrombocytopenic rabbits (Evatt et al., Blood, vol.54, pp.377-388, 1979; Hill and Levin, Exp. Hematol., vol.14, pp.752-759, 1986).

After purification of this factor was continued, was detected the average mol. the mass was 40000-46000 determined by gel-filtration (Kllr et al., Exp. Hematol., vol. 16, pp.262-267, 1988; Hill et al., Exp. Hematol., vol.20, pp. 354-360, 1992). Because IL-6 activity was not detected in plasma of rabbits with severe acute thrombocytopenia induced by the introduction of antisera against platelets, it has been suggested that this TRO-like activity was determined by factors other than IL-6 (Hill et al., ld, vol. 80, pp. 346-351, 1992).

Tayrien Rosenberg also cleaned the factor, with an average mol. the weight of 15000, from plasma thrombocytopenic rabbits and culture supernatant SOME cells, which stimulates the formation of platelet factor 4 in line megakaryocytic cells of rats, but not reported information on its structure (Tayrien and Rosenberg, J. Biol., vol.262, pp. 3262-3268, 1987).

In addition, Nakeff found Honey-CSF activity in serum thrombocytopenic mice, induced by the introduction of antisera against platelets (Nakeff, "Experimental mtology Today" ed. by Baum and Ledney, Springer-Verlag, NY, pp. 111-123, 1977). On the other hand, sera from thrombocytopenic rabbits increased the maturation of megakaryocytes (Keller et al., Exp. hematol., vol.16, pp.262-267, 1988; Hill et al., Exp. Hematol., vol.17, pp.903-907, 1989) and stimulated morphological change of megakaryocytes into platelets (Leven and forth in the plasma of rats, become thrombocytopenic under the influence of sublethal irradiation of the whole body (Miura et al. , Blood, vol. 63, pp. 1060-1066, 1984), and suggested that the induction of Honey-CSF activity in vivo is associated with a decrease in megakaryocytes, but not with a decrease in platelets, since this activity was not altered by the transfusion of platelets (MIGA et al. , Exp.Hematol vol. 16, pp. 139-144, 1988), Mazur and South found ef-CSF activity in the serum of irradiated sublethal dogs and reported that this factor has an average mol. a lot 175000 when measured by gel-filtration (azur and South, Exp.Hematol., vol. 13, pp. 1164-1172, 1985). In addition to the above, the factors obtained from the serum, plasma and urine was reported by other researchers, for example, Straneva et al. , (Straneva et al., Exp. Hematol., vol.15, pp. 657-663, 1987).

Thus, as described above, different activity, stimulating megakaryocytopoiesis and trombozitopoez, were detected in a biological sample obtained from thrombocytopenic patients and animals, but the selection of these factors, their biochemical and biological identification and determination of their properties have not been achieved due to their extremely small quantities in natural sources such as blood and urine.

A brief statement of the substance of the invention

The objectives of the AET activity of stimulating or increasing the formation of platelets in vivo and/or enhance proliferation and differentiation of progenitor cells megakaryocytes (hereinafter referred to as "TPO activity"), and isolation of the gene encoding the protein TRO, and providing ways to obtain this protein in a homogeneous form and in large quantities by recombinant DNA technology. Success in achieving these goals will lead to the replacement or reduction of the frequency used in the present transfusion of platelets, and this new protein is also used for the treatment and diagnosis of disorders associated with platelets.

Thus, this invention relates to (i) purified and selected DNA sequence that encodes a protein with TPO activity, selected from the group consisting of:

(a) the DNA sequence shown in SEQ ID 194, 195 and 196, or their complementary circuits, and

(b) DNA sequences for hybridization under stringent conditions with the DNA sequences of p.(a), or their fragments, and

(C) DNA sequences that were hybridizations with DNA sequences of paragraphs (a) and (b), if not for the degeneracy of the genetic code,

(ii) method of obtaining protein with TPO activity, providing stage:

growing under suitable conditions, the power prokaryotic or eukaryotic host cells transformed for transfected with the indicated DNA sequence still the military expression of a specified DNA sequence, and

(iii) the protein product obtained by expression in a prokaryotic or eukaryotic host cell of a specified DNA sequence.

In addition, this invention relates to a pharmaceutical composition containing an effective amount of the protein with TPO activity, and treatment-related platelet disorders, particularly thrombocytopenia, providing for the introduction of this protein in patients with such disorders.

Brief description of drawings

Fig. 1 shows a Sephacryl S-200HR gel filtration chromatography on Phenyl Sepharose 6 FF/LS F2, obtained from XRP.

Fig.2 shows Capcell Pak C1 chromatography with reversed phase UMC-pack CN-AP SRW-active fraction obtained from the sample of low-molecular TRO (Sephacryl S-200HR F3) XRP.

Fig. 3 shows the analysis in PAG-ordinator (SDS-PAGE) Capcell Cancer C1 TRO-active fraction (PA) from the sample of low-molecular TRO XRP.

Fig.4 shows the peptide maps on C18 HPLC with reversed phase TRO rat, isolated by electrophoresis in SDS page-ordinator (SDS-PAGE). Peptide fragments were obtained by systematic hydrolysis using three proteases.

Fig. 5 shows TRO activity obtained from XRP in a rat test system CFU-MK.

the culture supernatant COSI cells, which was introduced pEF18S-A2 in rat test system CFU-MK.

Fig. 8 shows TPO activity in the culture supernatant COS1 cells, which was introduced in pEF18S-HL34, rat test system CFU-MK.

Fig. 9 shows TPO activity in the culture supernatant COS1 cells, which was introduced in pHT1-231, rat test system CFU-MK.

Fig.10A shows TPO activity in the culture supernatant COS1 cells, which was introduced in pHTF1 in a rat test system CFU-MK.

Fig.10b shows TPO activity in the culture supernatant COS1 cells, which was introduced in pHTF1, in the test system M-e.

Fig. 11 shows the restriction map of clone GT1 and design pHGT1 pEFHGTE.

(E:EcoR1, H: Hind 111, S; Sal1)

Fig.12A shows TPO activity in the culture supernatant COS1 cells, which was introduced in pEFHGTE, rat test system CFU-MK.

Fig.12b shows TPO activity in the culture supernatant COS1 cells, which was introduced in pEFHFTE, in the test system M-e.

Fig.13A shows TPO activity in the culture supernatant COS1 cells, which was introduced in pHT1-211 1, pHT1-191 1 or pHT1-171 2, in rat test system CFU-MK.

Fig.13b shows TPO activity in the culture su is About activity in the culture supernatant COS1 cells, which was introduced in pHT1-211 1, pHT1-191 1, pHT1-171 2 or pHT1-163 2, in the test system M-e.

Fig. 15 is a chromatogram obtained by chromatography with reversed phase (Vydac column) for treatment of human solid radwaste from the culture supernatant of Cho cells, which was introduced in the vector pDEF202-hTPO-P1, Express tropic TRO.

Fig. 16 is a photograph showing the separation in SDS-PAGE (SDS page-ordinator) human TPO, purified from the culture supernatant of Cho cells, which were introduced pDEF2020 hTPO-P1 and gave expressed TRO.

Fig. 17 is a chromatogram obtained by chromatography with reversed phase purification of human TPO from E. coli, which was introduced pCFM536/h 6T(1-163) for the expression of TPO.

Fig. 18 is a photograph showing the separation in SDS-PAGE (SDS page-ordinator) variant of human TPO (1-163) allocated purified from E. Li, which was introduced F536/h6 (1-163) and allowed to be expressed TRO.

Fig. figure 19 shows the elution hO163 on a column of Superdex 75 rd in connection with cleaning hT 163 from the culture supernatant as the source material obtained by transfection of expressing the human TPO plasmids pDEF202 hTO163 in Cho cells. The amount of protein was determined at 220 N. the hO163 from the culture supernatant as the source material, obtained by transfection of expressing the human TPO plasmids pDEF202-hTPO163 in Cho cells. Protein hTPO163 were stained with silver gel.

Fig.21 shows the structure expressing vector pSMT201.

Fig. 22 shows a graph depicting TPO activity, a specific test M-e, in the culture supernatant of COS7 cells, which were injected GL-TPO, 3/TRO or 09/TRO and then took place in them the expression.

Fig. 23 is a graph depicting TPO activity, a specific test, M-07e, in the supernatant of cultures COS7 cells that were injected derived by insertion or a deletion of the human TPO, and then spent the expression.

Fig.24 shows an increased number of platelets in mice, which were injected TRO using intravenous and subcutaneous injection.

Fig.25 shows a dose-dependent increase in the number of platelets in mice after subcutaneous injection TRO.

Fig. 26 shows induced TRO increase in the number of platelets after treatment of mice with 5-FU to induce thrombocytopenia.

Fig. 27 shows induced TRO increase in the number of platelets after treatment of mice with hydrochloride of nimustine to induce thrombocytopenia.

Fig. 29 shows induced TRO increase in the number of platelets after x-ray irradiation of mice to induce thrombocytopenia.

Fig. 30 shows a dose-dependent increase in the number of platelets after the introduction of shortened TRO (amino acids 1-163 in SEQ 1D 6).

Fig. 31 shows an increase in the number of platelets after the introduction of shortened TRO (amino acids 1-63 in SEQ 1D 6) after the introduction of the hydrochloride of nimustine to induce thrombocytopenia.

Fig.32 shows that increasing concentrations of Mr1-X, added to the culture system of human megakaryocytes, leading to increased blocking the development of megakaryocytes.

Fig. 33 is TRO activity defined in the test M-e derived SRW [Met-2, Lys-1Ala1, Val3, Arg133]- TPO(1-163), [Met-2, Lys-1Ala1, Val3Pro148] TPO(1-163) [Met-2, Lys-1Ala1, Val3, Arg115] TPO(1-163), expressed in E. coli.

Fig.34 describes TRO activity defined in the test, M-07e, derivatives SRW [Met-2, Lys-1Ala1, Val3, Arg129]- TPO(1-163), [Met-2, Lys-1Ala1, Val3, Arg143] TPO(1-163), [Met-2, Lys-1P>, Lys-1Ala1, Val3, Arg59] TPO(1-163), expressed in E. coli.

A detailed description of the invention

This invention specifically provided polypeptides of thrombopoetin (SRW) having the biological activity of specific stimulus or increasing the formation of platelets containing amino acid sequence 1-332 SEQ 1D 6, or their derivatives. Cited as illustrations polypeptides include polypeptides that comprise amino acid sequences 1-163 SEQ 1D 6, 1-232 SEQ 1D 6, 1-151 SEQ 1D 6, the Mature amino acid sequence of SEQ 1D 2, 4 and 6, including polypeptides having deleteregvalue 1-6 amino acids NH2-end. Additional cited to illustrate the polypeptides of the present invention include [Thr33, Thr333Ser334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr244Ala345, Gly346, Val347, His348, His349, His350, His351, His352, His353, ] TPO, [Asn25, Lys231, Thr333Ser334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr344Ala345, Gly346, Val347 the e polypeptides of this invention include derivatives TPO polypeptides [His33] TPO(1-163), [AGD117] TRO(1-163), [Gly116]TPO (1-163), [His33, Thr33', RHS34] TPO(1-163), [His33Ala33'Pro34] TPO(1-163), His33, Gly33'Pro34Ser38] TPO(1-163), [Gly116, Asn116', Arg177] TPO(1-163), [Gly116Ala116', Arg177] TPO(1-163), [Gly116, Gly116', Arg117] -TPO(1-163), [Ala1, Val3, Arg129] TPO(1-163), [Ala1, Val3, Arg133] -TPO(1-163), [Ala1, Val3, Arg143] TPO(1-163), [Ala1, Val3, Leu82] -TPO(1-163), [Ala1, Val3, Leu146] TPO(1-163), [Ala1, Val3Pro148] -TPO(1-163), [Ala1, Val3, AGD59] TRO(1-163) and [Ala1, Val3, Arg115] -TPO(1-163).

TPO polypeptides of this invention also include polypeptides that are covalently bound to the polymer, preferably polyethylene glycol. In addition, TPO polypeptides of the invention may include amino acids [Met-2-Lys-1] , [Met-1] or [Gly-1]. The invention provides DNA that encode TPO polypeptide and derivatives described above, and these DNA provided in the form of cDNA, genomic DNA and synthetic DNA.

The invention also provides methods of obtaining TPO polypeptide described above, providing the stage of expressely expressed TPO polypeptide is et-2-Lys-1-polypeptide, these methods include the stage of detachment of et-1-Lys-1from the selected TPO polypeptide.

The invention also provides methods of obtaining TRO polypeptides, such as polypeptides, fused with glutathione-S-transferase (GST). DNA encoding amino-terminal GST polypeptide, peptide, binding thrombin, and TPO polypeptide is introduced into a suitable host, produce fused polypeptide and GST-part is removed by treatment with thrombin. Received TPO polypeptides have [Gly-1] structure.

Additionally provided prokaryotic and eukaryotic host cell transformed or transfetsirovannyh a DNA sequence according to the invention so that the host cell can Express a polypeptide having the biological activity of stimulating or increasing the formation of platelets.

The pharmaceutical compositions of this invention contain an effective amount of waste polypeptide or its derivative in combination with a pharmaceutically acceptable carrier and valid for use in the treatment associated with platelet disorders, in particular, for the treatment of thrombocytopenia, such as those caused by chemotherapy, the beam is.

Finally, this invention provides antibodies that specifically immunodiagnosis with TPO polypeptides and their derivatives, described above. Such antibodies are applicable in the methods for isolating and quantifying TPO polypeptides of the invention.

According to this invention, provided a new DNA sequence encoding a protein having TPO activity (hereinafter referred to as the DNA sequence of the present invention). The DNA sequence of the present invention contains a DNA sequence that encodes the amino acid sequence of the DNA which encodes the amino acid sequence shown in SEQ 1D 2, 4 or 6 of the attached list of sequences.

Also the DNA sequence of the present invention is called the DNA sequence encoding a partially modified by substitution, deletion, insertion or addition) version of the above amino acid sequence shown in SEQ 1D 2, 4 or 6, provided that such modifications do not violate the TRO activity. I.e., the DNA sequence encoding the derived TRO, also included in this invention.

In other words, the DNA sequence of this invention vkluchaet are amino acid sequences, shown in SEQ 1D 2, 4 or 6. The phrase "amino acid sequences are essentially amino acid sequences shown in SEQ 1D 2, 4 or 6" as applied here means that these amino acid sequences include the sequence represented by SEQ 1D 2, 4 or 6, and sequence represented by SEQ 1D 2, 4 or 6, which have a partial modification, such as substitution, deletion, insertion, addition, and so on, provided that such modifications to violate the TRO activity.

Further, the DNA sequence of this invention consists essentially of a DNA sequence that encodes a protein with TPO activity.

The term "DNA sequence encoding amino acid sequence" includes all DNA sequences that can be degeneracy in nucleotide sequences.

The DNA sequence of this invention also includes the following sequence:

(a) the DNA sequence represented by SEQ 1D 7, 194, 195 and 196, or complementary to them chain,

(C) DNA sequences for hybridization under stringent conditions with the DNA sequences defined in (a), or their fragments, or

(the ability of the genetic code.

In other words, the DNA sequence of this invention also includes the following sequence:

(a) a DNA sequence that is integrated into the vector pEF18S-A2 (Deposit FERM BP-4565) to be included in the strain E. coli DH5., vector pHT1-231 (Deposit FER BP-4564) included in the strain E. coli DH5. vector pHTGI (Deposit FER BP-4617) included in the strain E. coli DH5. vector GT1 Deposit (FERM BP-4616) included in the strain E. coli DH5., and encodes the amino acid sequence of the protein having TPO activity, or

(C) DNA sequences which hybridize (under strict conditions) with the DNA sequences described in (a), or their fragments, and encode the amino acid sequence of a protein having TPO activity.

In the application here, representative "stringent" hybridization conditions are those conditions that applied in the examples by PCR amplification of the DNA of this invention using degenerate and/or unique renogotiated primers (probes). Cm. also, for example, chapters 11 and 14 Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) Unit 2.10 Current Protocols in Molecular Biology, Ausubel et al., eds. Current Protocols, USA, 1993).

Also the sequence of the present invention is a DNA sequence, which is inoculates sequence, presents SEQ 1D N 6.

Such DNA sequences may contain also the site of cleavage by the restriction enzyme and/or additional DNA sequence at the site of initiation, termination or intermediate site, which facilitate construction of readily expressed vectors. When using the owner, which is not a mammal, can be included preferred codon for gene expression in this host.

An example of the DNA sequence of the present invention is a cDNA molecule, obtained by the selection of mRNA from mammalian cells, including human, with subsequent screening of this cDNA in the usual way from a cDNA library obtained in a known manner. Sources mRNA in this case may be cells derived from rat hepatocytes cell line McA-R N, NTS cells, N-E cells, rat liver, kidney, brain and small intestine of the rat, human liver, etc.

Another example of the DNA sequence of the present invention is a molecule in the genomic DNA, which can be obtained by screening it in the usual way from a genomic library obtained in a known manner from mammalian cells, including human. Sources of genomic DNA in this case may be the drug is otvagnoe TRO, can be obtained from the obtained in the manner described cDNA sequence that encodes a protein with TPO activity by modification of this cDNA using site-directed mutagenesis, partial modification thereby of the corresponding amino acid sequence.

After clarifying the amino acid sequence or DNA sequence of a protein having TPO activity, in this invention, the DNA sequence encoding a slightly modified amino acid sequence, can be easily obtained by chemical synthesis.

The DNA sequence of this invention is a valuable material for large-scale obtain a protein having TPO activity, using various methods of recombinant DNA.

The DNA sequence of the present invention is applicable also as a labeled probe for isolation of the gene encoding related TRO protein, as well as cDNA and genomic DNA encoding SRW other mammalian species. It is applicable in gene therapy of human and other mammalian species. In addition, the DNA sequence of the present invention is applicable for the development of transgenic species melchorita that can the pp. 809-814, 1983).

This invention also provides a vector which integrates the above DNA sequence encoding a protein having TPO activity, the host cell, transformed with this vector, and a method of obtaining a protein with TPO activity involving the cultivation of the host cells and the separation and purification of the expressed protein having TPO activity.

Examples of host cells applicable in this case, are prokaryotic cells, such as cells of E. Li, etc., and eukaryotic cells such as yeast, insects, mammals, etc., Illustrative examples of mammalian cells are COS cells, cells of the Chinese hamster ovary (SEO), C-127 cells, kidney cells baby hamster (KSS), etc., Illustrative examples of yeast is Baker's yeast (Saccharomyces cerevisiae), assimilating methanol yeast (Pichia pastoris) etc. Illustrative examples of the insect cells are cultured cells of the silkworm, etc.

With regard to vectors for use in transforming these host cells, to transform cells of E. coli can be taken RX (Shimatake, H. and M. Rosenberg, Nature, 292, 128-132, 1981), pTrC99A (Amann E. et al., Gene, 69, 301-315, 1988), etc. To transform the-SR296 (Takebe et al. , Mol. Cell. Biol. 8, 466-472, 1988) or similar To yeast cells can be applied G-1 (M. Schena and Yamamoto, K. R., Science, 241, 965-967, 1988) or etc., For the transformation of the silkworm can be applied transducerless vector for recombinant viral constructs, for example, RAS (Luckow et al., Bio/Technology, 6, 47-55, 1988).

If required, each of these vectors may contain the beginning of replication, marker (markers) selection, promoter, etc., and is also the site of RNA splicing, polyadenylation signal, etc., in the case of vectors for use in eukaryotic cells.

As for the start replication, vectors for mammalian cells, you can apply the sequence obtained, for example, SV40, adenovirus, virus, bovine papilloma or etc In vectors to cells of E. coli can be applied sequence derived from ColE1, R factor F factor or etc In vectors for yeast cells can be applied to the sequence obtained from 2 μm DNA, ARS1 or etc.

As promoters for gene expression, vectors for mammalian cells can be applied promoters obtained from, for example, retrovirus, virus polyoma, adenovirus, SV40, etc., a Promoter derived from bacteriophage , for example, trp, lpp, lac or tac promoter, can p is x's yeast, and OH promoter or etc in vectors for assimilating methanol yeast. A promoter derived from a nuclear polyhedrosis virus, can be used in vectors for silkworms.

Typical examples of breeding markers applicable to vectors for mammalian cells, is the gene of resistance to neomycin (peo), gene timedancing (TS), gene digidrofolatreduktazy (DHFR) gene xanthine-guaninephosphoribosyltransferase E. coli (ECOgpt), etc., Illustrative examples of breeding markers applicable to vectors for E. coli cells, is the gene for resistance to kanamycin resistance gene, the ampicillin gene of resistance to tetracycline, etc., and markers for yeast cells are genes Leu2, Trp1, Ura3, etc.

Getting protein with TPO activity by using appropriate combinations of host-vector systems may be performed by transforming suitable host cells with recombinant DNA obtained by integration of the gene of the present invention in a suitable site in the specified vector, culturing the resulting transformant with subsequent separation and purification of the target polypeptide obtained from cells or culture medium or filtrate. Commonly used tools and procedures can and shall sledovatelnot can be modified or replaced with a signal sequence, derived from another protein, in order to obtain a homogeneous N-end of the expressed product. The homogenization of the N-end can be performed by a modification (replacing or adding) amino acid residues in the N-end or near it. In the case expression using E. coli as a host cell, for example, the lysine residue may be added in addition to the residue of methionine.

A new protein of the present invention, having TPO activity (hereinafter referred to as "protein of this invention"), includes proteins, each of which contains the amino acid sequence shown in SEQ 1D 2, 4 or 6. Derivatives TRO, amino acid sequence which is partially modified (substitution, deletion, insertion or addition), also included in this invention, provided that TPO activity is not disturbed by such modification.

In other words, the protein of the present invention includes protein molecules, amino acid sequences which are essentially the amino acid sequences shown in SEQ 1D 2, 4 or 6.

The expression "amino acid sequence are essentially the amino acid sequences shown in (or of) SEQ is eljnosti, shown in SEQ 1D 2, 4 or 6, and the sequence shown in SEQ 2, 4 or 6, which have a partial modification, such as substitution, deletion, insertion, addition, and so on, provided that such modifications do not violate the TRO activity.

The protein of the present invention includes a protein containing provisions 7-151 amino acid sequence shown in SEQ 1D 6, and having TPO activity. Also protein of the present invention is a protein that has a solid radwaste activity and contains provisions 1-163 amino acid sequence represented by SEQ 1D 6.

Examples of other SRW derivatives of this invention include derivative, stability and life expectancy which in vivo have been improved by modification of amino acid substitution, deletion, insertion or addition), a derivative in which at least one potential glycosylation has been modified by deletion or addition of, a derivative in which at least one cysteine residue was deleterows or replaced by another amino acid residue (for example, the residue of alanine or serine).

Preferably, the protein of the present invention differs in that it is separated and purified from host cells transformed rivers of the m synthesis.

When intracellular expression is carried out with the use of bacteria, such as E. coli as a host, get protein, in which the initiating methionine residue added to the N-terminal side of the protein molecule having TPO activity, which is also included in this invention. Depending on the host protein obtained with TPO activity may be glycosylated or deglycosylated and each of these cases are included in this invention.

The protein of the present invention also includes the naturally occurring TRO-active proteins, purified and isolated from natural sources, such as the environment, culture of cells with TPO activity, urine, serum or plasma of man.

Cleaning method SRW from such natural sources are also included in this invention. This cleaning can be performed by applying one or a combination of commonly used stages of purification of proteins, such as ion exchange chromatography, affinity chromatography with the use of lectins, affinity chromatography with a triazine dye, hydrophobic chromatography, gel chromatography, chromatography with reversed phase, affinity chromatography with heparin chromatography with sulfatrim chelates metals, preparative electrophoresis, gel electrophoresis with isoelectric focusing, etc., the cleaning Process, in which use certain methods in combination with the use of physico-chemical properties of the solid, which can be derived from the examples of this application are also included in this invention. In addition, you can also use affinity chromatography using antibodies able to recognize TRO. Further, it was found that the TRO is a ligand for l (de Sauvage et al., Nature, 369; 533-538 (1984); Bartley et al., Cell 77: 1117-1124 (1994); Kaushansky et al. , Nature, 369: 565-568 (1984), in which the solid can be purified using affinity gel column, which is associated l. More specifically, an example of such a column is l's column, prepared by linking resin with the extracellular region of the Mpl (l-X), obtained by means of recombinant DNA, using Cho cells as the host (Bartley et al., Supra).

As described herein, polypeptides TRO of this invention are distinguished further by the ability to communicate with l receptor and specifically with its extracellular (soluble) domain.

This invention also includes a protein encoded by a part of DNA that is complementary to the coding protein chain human cDNA or the amontan et al. (Nucl. Acids Res., vol.12, pp.5049-5059, 1984).

Also included in this invention the protein of the present invention, which marked detektivami marker, for example, 1251or bioterrorism, ensuring reagent applicable for the detection and quantitative determination TRO or expressing the receptor for TPO cells in solid samples, such as tissue and fluid samples such as blood, urine, etc.

Biotinylated protein of the present invention is applicable in the case of its binding to the immobilized streptavidin removal of megakaryocytes from bone marrow during autologous bone marrow transplantation. It is also applicable in the case of its binding to the immobilized streptavidin concentrate autologous or allogeneic megakaryocytic cells during autologous or allogeneic bone marrow transplantation. Conjugate SRW with a toxin, such as ricin, diphtheria toxin, or etc., with a radioactive isotope is applicable in cancer therapy and conditioning for bone marrow transplantation.

This invention also provides a material of nucleic acids, which is applicable when tagging detektivami marker, including radioactive marker or nania provisions of the human gene SRW and/or related families of genes on the chromosomal map. This material is also applicable to evidence of violations in the gene SRW person at the DNA level, and it can be used as a genetic marker to confirm violations of the adjacent genes.

This invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the protein of the present invention together with a useful and effective diluent, an antiseptic agent, solubilizers agent, emulsifier, adjuvant and/or carrier. The term "therapeutically effective amount" as applied here means an amount that provides a therapeutic effect for the stated conditions and ways of introduction and conditioning. Such a composition is used in the form of liquid, lyophilized or dried drug. It contains a diluent selected from different buffers (Tris-HCl, acetate buffer and phosphate buffer, for example) with different pH and ionic strength, which prevents the surface absorption of additives such as albumin or gelatin, surfactant, such as Tween 20, Tween 80, Pluronic F68 or salt of bile acid, solubilizers agent, such as glycerin or polyethylene glycol, an antioxidant, takepart or paraben, and the carrier or agent toychest, such as lactose or mannitol. Also applicable composition, providing for covalent binding of the protein of the present invention with the polymer, such as polyethylene glycol, chelating proteins with metal ions, the inclusion of protein in granular preparation or on the surface consisting of polymeric compounds such as polylactic acid, polyglycolic acid or hydrogel, or the inclusion of this beam in liposomes, microemulsions, micelles, single-layer or multilayer vesicles, "shade" of red blood cells or spheroplasts. This composition will be affected by the physical conditions, solubility, stability, rate of release in vivo and clearance in vivo TPO.

Select a song performed depending on the physical and chemical properties of the applied TRO-active protein. This invention also provides a granular composition, in which the granules coated with the polymer, such as poloxamer or poloxamine, and TPO, which is associated with antibodies to tissue-specific receptors, ligands or antigens or tissue-specific ligands of the receptors. Other examples of compositions of this invention are compositions in the form of granules, which are samih introduction, such as parenteral, pulmonary, transnasal and oral administration.

The pharmaceutical composition containing the protein of the present invention, may be injected several times a day, usually in the amount of 0.05 μg - 1 mg/kg (based on protein TPO) of body weight depending on the condition and sex of the patient, the route of administration, etc.

The pharmaceutical composition containing the protein of the present invention may be administered in a dose of 25000-500000 active ingredient (in units of relative activity obtained with the help of test M-e, which will be described later) per kg of body weight once or several times per day for 1-7 days per week depending on the symptoms, sex of the patient and the method of administration.

The authors of this proposal have confirmed that in relation to the C-terminal side of human TPO can be noted that the activity is saved, when amino acid residues up to 152-th amino acid in the amino acid sequence represented in SEQ 1D 6, deleterow, and that the N-terminal side, activity is maintained even if the amino acid residues up to the 6th amino acid residue sequence deleterows.

Thus, a protein having TPO activity, soderjanii, a deletion, insertion or addition) in other parts, may preferably be used as the effective ingredient of this study as well as unmodified protein. Preferred derivatives TRO is derived, having the amino acid sequence 1-163 sequence SEQ 1D 6.

A composition comprising at least one additional hematopoietic factors such as EPO, G-CSF, GM-CSF, M-CSL, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF, in addition to the protein of the present invention, also included in this invention.

The protein of the present invention is applicable for the treatment of various thrombocytopenic disorders, either separately, or in combination with other hematopoietic factors. Examples of other additional hematopoietic factors are EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF.

There are many diseases characterized associated with platelet disorders such as thrombocytopenia, caused by disturbances in the formation of platelets or reducing the life of the platelet (caused by destruction or consumption of platelets), is the service of platelets in thrombocytopenic patient, the disease which is caused by congenital gipoplasticheskaya anemia with pancytopenia and other anomalies (Fanconi syndrome) or gipoplasticheskaya anemia caused by chemotherapy or radiation, myelodysplastic syndrome, acute myelocytes leukemia, aplastic crisis after bone marrow transplantation. Thrombocytopenia caused by reduction of the lifespan of the platelets or megakaryocytes, includes idiopathic thrombocytopenic purple, gipoplasticheskaya anemia, acquired immunodeficiency syndrome (AIDS), a syndrome of diffuse intravascular coagulation and thrombotic thrombocytopenia. In addition, protein can also be applied in the case of autotransfusion of blood platelets, where TRO is administered to the patient before surgery to increase the number of native platelets of the patient and, therefore, increased the platelet count is used as the transfused platelets at the time of the operation.

Another application of the protein of this invention is the treatment of diseases associated with a temporary reduction or destruction of platelets caused by other chemical or pharmaceutical drugs or medical treatments. TRO m this invention relates to antibodies, specific for TPO. The protein of this invention can be used as antigens and corresponding antibodies include monoclonal and polyclonal antibodies and chimeric antibodies, namely, "recombinant antibodies, obtained by conventional methods. To obtain such antibodies against TPO can be used by human TPO as antigen or, alternatively, a partial peptide of human TPO can be used as antigen. When using antibodies to the antigen such a peptide epitope can be determined by specifications of the antigenic region. On the other hand, the use of antibodies to antigenic protein(SRW) antigenic region may be clarified by analysis of the epitope of the antibody. In such cases, these antibodies, each of which has been elucidated thus spitup, can be used for the separation, detection, quantification and purification of various TRO, which differ in their properties, for example, the type of sugar added, the length of peptide chains, etc.

Peptide TPO containing selected amino acid sequence, synthesize and connect with a suitable protein carrier, for example, avego antigen. Alternatively, the peptide-type peptide with multiple antigens (MAP) receive for use as an antigen according to the method of Tam (Proc.Natl. Acad. Sci. (USA) 85:5409-5423, 1983). Then thus prepared antigen together with an adjuvant or etc., used for immunization of mammals, birds and so on, which are usually used for the production of antibodies, such as rabbits, mice, rats, goats, sheep, chickens, hamsters, horses, Guinea pigs, etc. From immunized thus the animal was received his anticavity and cells of polyclonal antibodies. When using a peptide as an antigen, the epitope may be determined by the specification of the antigenic region. In this case, the areas of different molecules SRW with different mol. mass is subjected to screening and identify with the help of immunological engineering. For example, you can skanirovat and identify the area with a deletion of the peptide. In addition, the gene of the antibody or part of this gene clone of cells expressing the target antibody, to obtain a molecule antibodies, which was expressed with the use of genetic engineering.

Compared with the polyclonal antibody that contains a variety of antibodies specific to different ant is on the antigen. Specific for TPO antibody is applicable to improve the selectivity and specificity of diagnosis, based on the reaction of antigen-antibody, and an analytical test method, as well as in carrying out the separation and purification of solid radwaste. Additionally, such antibodies can be used to neutralize or remove TRO from the serum. Monoclonal antibodies are also applicable for the detection and quantitative determination TRO, for example, serum whole blood.

Antibody against TRO person present invention can be used as a ligand in affinity chromatography for the purification and selection TRO person. For fixation of the antibody so that it can be used in affinity chromatography, can be used any conventional fixing of various enzymes. For example, we apply the method using media Nr - activated Sepharose 4B (produced by Pharmacia Fine Chemicals Co. or similar To clean TRO person using a fixed antibodies against TPO person fixed antibody loaded into the column and containing TRO human fluid is passed through this column. In this operation, a large amount of waste of the person is adsorbed on the media in the column. the new acid, dioxane, ethylene glycol, jatropha salt, guanidine hydrochloride, urea, etc. By similar elution solvent elute highly TRO person.

The antibody of the present invention can be applied to determine TRO human quantitative immunochemical analysis, in particular, enzyme linked immunosorbent assay, carried out by the method of solid-phase sandwich.

The advantage of monoclonal antibodies is that they can be produced hybridoma cells in an environment that does not contain any other molecules of immunoglobulins. Monoclonal antibodies can be obtained from the culture of supernatants hybridoma cells or from mouse ascites induced intraperitoneally injection of hybridoma cells. Hybridoma method, a source revealed Kohler Milstein (Eur.J. Immunol. 6:511-519, 1976), can be widely used to obtain lines of hybrid cells, which have high levels of monoclonal antibodies to specific antigens.

For selection of the target antibodies of the thus obtained containing the antibody of the material, such as anticavity and so on, can be combined in one or several stages, which are usually used for cleaning Bel the spruce-chromatography gel chromatography with a fixed anti-immunoglobulin, etc., as well as cation exchange chromatography, anion exchange chromatography, affinity chromatography with the use of lectins, adsorption chromatography with dye, hydrophobic chromatography, helpanimals chromatography, chromatography with reversed phase, hydroxyl-Apatity chromatography, fluorine-Apatity chromatography, chromatography with the use of metal chelates, chromatography isoelectric focusing, distribution chromatography, electrophoresis with isoelectric focusing, and so on). In addition to these methods may be also used a method of affinity purification using antigen, which serves gel media or membrane that is chemically attached to the protein TRO person or peptide containing region of the antigen or the part of the area, i.e., a molecule that is able to learn the target antibody to the media or membrane, thus obtained, add containing the antibody of the material, so that the target antibody is adsorbed on the media or membrane. Then adsorbed thereby elute antibody and extracted with suitable conditions.

This invention also allows you to apply and is of FL. For example, can be applied to methods of homologous recombination in vitro and ex vivo transformation of host cells with the aim of ensuring expression or enhanced expression of polypeptides. Preferred host cells are human cells (e.g., liver, bone marrow, etc.,), which impose a promoter or enhancing sequence, using flanking sequences that are homologous to the target area in the cellular genome, which results in or increases the expression of TPO polypeptide. See, for example, in U. S. Letters Patent 5 272 071, published PCT WO 90/14092, WO 91/06666, WO 91/09955.

This invention also provides methods of obtaining TRO polypeptide described above, providing the stage of expression of the polypeptide encoded by the DNA of the present invention, in a suitable host and select the TPO polypeptide. If expressed TRO the polypeptide is Met-2- Lys-1polypeptide, such methods may provide further stage of detachment Met-2- Lys-1from the selected TPO polypeptide.

Also provided methods of obtaining TRO polypeptides having [Gly-1] the structure, providing the stage of introduction into a suitable host cell is libated, moreover, GST and TPO polypeptide coding sequences separated DNA that encodes a polypeptide that recognizes thrombin, separation of the product of expression of GST-TPO and processing of the expressed polypeptide with thrombin to remove amino acids GST. Received TPO polypeptides have [Gly-1] structure.

The invention is described hereinafter in detail.

(A) Purification of rat TRO, the analysis of partial amino acid sequences of purified rat TRO and analysis of biological characteristics of purified rat TRO.

The authors of the present invention first tried to remove the protein (rat TRO), which has an activity of enhancing the proliferation and differentiation of rat CFU-MK. In this study, purification were made many mistakes in cleaning procedures, such as selection of different natural food sources, selection of gels for chromatography and separations. As a result, the authors were able to purify a protein having TPO activity of blood plasma thrombocytopenic rats, induced by irradiation of x-rays or-rays, using SRW activity as a marker in the test with rat CFU-MK, which will be described below in "Standard Example", and to determine what cteristic obtained from rat plasma TPO also defined in "Example 3".

The basics of procedures from cleaning rat TPO to determine the partial amino acid sequences of the purified protein set forth herein.

(i) a Sample of blood plasma was obtained from approximately 1100 thrombocytopenic rats induced by x-rays or-rays, and subjected to chromatography on Sephadex G-25, anion-exchange chromatography (Q-Sepharose FF) and lectinology chromatography (WGA-Agarose) in that order, getting adsorbed on WGA-Agarose TRO-active fraction.

(ii) Then the adsorbed TRO-active fraction was subjected to affinity chromatography with triazine dye (TSK AF-BLUE 650 MN), hydrophobic chromatography (Phenyl Sepharose 6 FF/LS) and gel chromatography (Sephacryl S-200 HR) in this sequence. Because TPO activity was divided into 4 peaks (F1 (fraction 1) as the high-molecular fraction, then F2, F3 and F4) by gel filtration on Sephacryl S-200 HR, each of the TRO-active fractions F2 and F3 was concentrated to obtain samples F2 SRW with high mol. mass and sample F3 with low-molecular TRO, which are used separately in subsequent stages of treatment.

(iii) a Sample F3 low-molecular TRO was subjected to preparative chromatography with reversed phase YMC-Pack PRO-TEIN-RP) chromatography with on the received thus TRO-active fraction was applied on SDS page-ordinator (SDS-PAGE) electrophoresis and TRO-active substance was extracted from the gel, the presence of TPO activity was detected in the lane corresponding to the average mole. masses of approximately 17000-19000 when non conditions.

(iv) Subsequently, all the SRW-active fraction was applied to a gel SDS-PAGe under non conditions and transferred to PVDF membrane. By performing a systematic limited enzymatic hydrolysis of protein to PVDF membrane to peptide fragments were determined partial amino acid sequence of rat protein TRO. Based on the information about the amino acid sequences of the two peptide fragments were cloned gene rat TRO.

(v) apart from this, sample F2 macromolecular TRO obtained using Sephacryl S-200 HR, subjected to purification in the same way, which was purified sample F3 low-molecular TRO. When the SRW-active fraction obtained the final chromatography with reversed phase (Capcell Pack C1), were applied to the gel SDS-PAGE and TRO-active substance was extracted from the gel, the presence of TPO activity was detected in the lane corresponding to the average mole. masses of approximately 17000-22000 when non conditions.

(C) Specialization producing TRO rat cells, obtaining mRNA and is designed and the and it was necessary to skanirovat organs or cells as a source of mRNA for use in cDNA cloning. In the TRO activity in various organs and supernatant cell cultures were skanirovaniya on the basis of biochemical and biological properties of rat obtained from plasma TRO. In the TRO activity, almost equal to the activity obtained from rat plasma TPO were found in the culture supernatant of cell lines of rat hepatocytes McA-RH8994, HTC and H4-11-E, as well as in the supernatant of culture of rat primary hepatocytes (example 4).

On the other hand, was constructed expressing vector pEF18S of 2 expressroute vectors RME and FOS (example 5). The construction of this vector was made possible easy cloning of cDNA with the use of high-expressing vector with multiple cloning sites, which can be used to integrate indels.

In addition to the above expressing vector, to construct cDNA libraries were mostly plasmid vectors rice and pBR and vectors based on phage .

ACI-R8994 the granulosa cells, homogenized with the addition of a solution of guanidine thiocyanate and experience which should also be phenol method (with hot phenol), method using acidic guanidine-phenol-chloroform, etc.

After separation of the poly (A)+RNA from total RNA using oligo-dT-immobilized latex particles, synthesized the first chain cDNA using reverse transcriptase using oligo - dT as a primer, to which was added a sequence uznanie restrictase Notl, followed by synthesis of the second chain cDNA using RNase H and DNA polymerase 1 E. coli. EcoRl was added to the thus obtained double-stranded cDNA obtained cDNA ligated with expressing vector mammalian cell pEF18S constructed in example 5, which was split Notl and EcoRl, and then legirovannoi thus cDNA was transformed into competent cells of strain D5 E. coli to construct a cDNA library (example 7).

(C) Obtaining (clone) the cDNA fragment of rat TRO using PCR

The DNA sequence deduced from a partial amino acid sequence of rat TRO, purified from the blood plasma of rats for the synthesis of degenerate primers used in polymerase chain reaction (R). Primers for use in R can also be obtained on the basis of a different amino acid sequence, h is imeneniya inosine. In addition, primers with a reduced degeneracy can be constructed by applying the codon highly applicable in rats (Wada et al., Nucleic Acids Res., vol. 18, p. 2367-2411, 1990).

When plasmid DNA was extracted from all parts of the cDNA library obtained as described above, and PCR was performed using the extracted DNA as template, discovered a band of approximately 330 p. N., which was then identified by sequencing of nucleotides as DNA fragment (A1 fragment) encoding part of the rat TPO (example 8).

(D) Screening of cDNA rat TRO using PCR, the cDNA sequence of rat TRO and confirmation TRO activity

The cDNA library obtained as described above were divided into pools, each of which contained approximately 10,000 clones, and were extracted plasmid DNA from each of 100 pools. When conducting R using each pool of plasmid DNA as template and primers, newly synthesized based on the nucleotide sequence of the A1 fragment, 3 pools were discovered bands that are considered specific. One of the 3 pools were divided into subpoly, each of which contained approximately 900 clones. Plasmid DNA was purified from 100 subpool Dulov divided into subpoly, each of which contained 40 clones, and finally each clone was skanirovali using PCR as described. The result was the selected clone pEF18S-A2, which, apparently, cDNA encodes rat TPO (examples 9 and 10).

When analyzing the nucleotide sequence of this clone was found that it encodes a partial amino acid sequence of the protein, purified from the blood plasma of rats, confirming, therefore, a strong likelihood that this clone contains a cDNA rat TPO (example 10).

When purification of plasmid DNA from clone pEF18S-A2, obtained as described above, and transfection in COS 1 cells found TPO activity in the culture supernatant of transfected cells. This confirmed that the clone pEF18S-A2 contains cDNA encoding rat TPO (example 11).

(E) Detection of mRNA TRO in various tissues of rats

The expression of mRNA TRO in rat tissues was analyzed using R. Specific expression found in brain, liver, the small intestine and kidney (example 12).

(F) Constructing a cDNA library of human

Based on the results of examples 4 and 12 liver was chosen as the source of tissue for cloning cDNA of human TPO. Subsequently, the cDNA library con is the number of vector as in the case of cDNA library rat, synthesized cDNA in a similar way, getting the library directionally cloned cDNA using restricted Not1 and EcoR1. Library, is obtained by introducing legirovannykh with vector cDNA in E. coli DH5, contained approximately 1200000 clones (example 13).

(G) Receiving (cloning) of cDNA fragment of the human TPO using PCR

Several primers for PCR were synthesized based on the nucleotide sequence of clone pEF18S-A2, coding cDNA of rat TRO. During the synthesis of cDNA using a commercial mRNA obtained from normal human liver and R using these primers and cDNA as template watched band of approximately 620 p. N. In the analysis of nucleotide sequences found that this clone contains a DNA fragment that has a homology of approximately 86% with rat cDNA TRO, confirming, thus, is a strong likelihood that this part of the gene encoding TRO person (example 14).

(H) Screening of cDNA TRO person using R, the cDNA sequence TRO person and confirm the TPO activity

A library of human cDNA, obtained as described above, amplified, separated into the crystals. When conducting PCR using each pool of molecules of plasmid DNA as template and primers, newly synthesized based on the nucleotide sequence of the fragment TRO person, obtained in example 14, it is possible bands found in 3 pools. One of these pools were divided subpoly, each of which contained 5000 clones, and a plasmid DNA was purified from each of the 90 subpool. When conducting R using each pool of plasmid DNA as template in the same way possible band was detected in 5 Subpolar. When dividing one of these pools on subpoly, each of which contained 250 clones, purification of plasmid DNA from each of the 90 subclones and conduct of possible PCR bands were detected in 3 subclones. When dividing one of these pools on subpoly, each of which contained 30 clones, purification of plasmid DNA from each of the 90 subpool and holding R possible bands were detected in 3 Subpolar. After that allocated 90 colonies from one of these subpool and plasmid DNA purified from each of these colonies were subjected R, finally getting a clone, named HL34 (example 15).

When analyzing the nucleotide sequence of the plasmid DNA from this clone was found that this clone contains the Noah plasmid DNA and transfection her in COS1 cells TPO activity was detected in the culture supernatant of transfected cells. Thus, it was confirmed that this plasmid clone contains a cDNA encoding a TRO person (example 17).

However, apparently, this cDNA is an artificial product of cloning, because this clone was not detected termination codon and at its 3'end was detected sequence that is similar to the poly(A) tail. Was subsequently constructed expressing the vector that encodes the amino acid sequence, except for the part corresponding sequence similar to the poly(A) tail. When the expression is designed vector in COS 1 cells TPO activity was detected in the obtained culture supernatant (example 18).

Using R was obtained DNA fragment at the 3'-terminal region TRO person to examine the structure of the full-size cDNA.

Determination of the nucleotide sequence of this fragment found that it partially overlaps with the cDNA, which is clone HL34 obtained in example 15. Also expected that the full-size cDNA TRO person can contain an open reading frame to encode a protein, consisting of 353 amino acids, including a signal sequence of 21 amino acids (example 19).

However, since the content of the coding TRO RNA person, apparently, is very low in normal human liver, as will be described further in example 15 (contents, calculated from the results of this example, was in the ratio of 1: 3000000), hybridization screening, which is used synthesized oligonucleotide or cDNA fragment SRAW rats or humans as a probe, it will be very difficult to perform because the number of clones or plaques that need to be processed, becomes very large and the sensitivity and specificity of the hybridization method are lower than the sensitivity and specificity of PCR method. In fact, the authors of this invention have conducted hybridization of colonies of two million KLA cDNA SRAW rats as a probe, but was unable to obtain the cDNA clone TRO person.

(I) Reconstruction of cDNA derived from normal human liver

Because the clone HL34 obtained in example 15, apparently, contains incomplete cDNA, a cDNA library was reconstructed using commercial product obtained from normal human liver poly (A)+RNA to obtain a complete cDNA TRO person. This library (hTPO-F1) obtained by the introduction legirovannoi with vector cDNA in E. coli DH5, contained 1,0106the transformants (example 20).

(J) Screening of the cDNA clone TRO, sequencing and expression of cDNA TRO person and confirm the TPO activity

The primers for PCR were synthesized based on the nucleotide sequence (SEQ 1D 3) partial cDNA obtained in example 14, and the nucleotide sequence (SEQ 1D 196) complete cDNA SRAW man, foretold in example 19.

A cDNA library of a human liver (hTPO-F1) constructed in example 20 was divided into 3 pool (pools 1-3). R was carried out using the plasmid DNA obtained from each pool as template and synthetic primers. As a result, when using plasmid DNA obtained from the pool 3 was amplified DNA fragment having about the ning using PCR, as explained above. In the amplification of DNA with the expected size was detected in 6 of 90 pools. Dividing one of these positive pools on subpoly, each of which contained 1000 clones, extraction of plasmid DNA and PCR reaction in the same way did not observe DNA amplification. Believed that this is due to the low extraction of plasmid DNA due to weaker growth target clone compared to the growth of other clones. Therefore, the original pool of 3 printed on cups 100L In with the same amount of inoculum that each Cup L grew 4100 colonies, and from each of the inoculated thus cups were given a Cup replica. When conducting R these DNA extracted from colonies that grew on this plate, the amplification of the expected bands were observed in one of the 100 subpool.

Each Cup of this subpool were prepared by two filter-replica and hybridization of colonies was performed using the labeled probe (fragment EcoR1/BamH1 plasmids F18S-L34). As a result, only one signal was positive. Colonies were collected from the original Cup and again inoculable on L In the Cup. DNA samples were obtained from 50 colonies that grew on this plate, and exposed them to R, finally getting a clone, named pHTF1 (example 21). In the screening of the cDNA clone used the OPEC raised the efficiency and sensitivity of the screening or reduced labor costs for screening. When determining the nucleotide sequence taken so clone F1 found that the clone pHTF1 had an open reading frame and amino acid sequence of the protein encoded by (potentially) this open reading frame, was identical with that deduced amino acid sequence (SEQ 1D 6) TRO person. The nucleotide sequence differed from deduced (SEQ 1D 196) at 3 positions, but these differences did not cause amino acid changes. Confirmed that the protein TRO person contains 353 amino acid residue, including the signal sequence of 21 amino acids. Upon receipt of plasmid DNA from the thus obtained clone pHTF1 and transfection her in COS 1 cells TPO activity was detected in the culture supernatant (example 23).

(K) Screening for chromosomal DNA TRO person using hybridization spots, sequencing and expression of the chromosomal DNA TRO person and confirm the TPO activity

Genomic library obtained from Professor T. Yamamoto ((Gene Research Center, Tohoku University) were sown on the Cup 18 NZY in that the amount of inoculum that one Cup contains 30000 phage particles, and from each of the thus prepared 18 cups polvani using PCR and purified. Using this purified fragment, labeled32R, as a probe was performed hybridization spots. The result received 13 positive spots. Spots were collected from the original cups and inoculable again on Cup NZYM in that the amount of inoculum that each Cup has formed 1000 spots. From each of the cups were prepared by two filter replicas for conducting hybridization spots under the conditions described above. The positive signals were detected in all filters 13 groups. One spot (the plaque) was extracted from each of the cups to get ragovoy DNA. Sample phase DNA thus obtained of the 13 clones were checked for the presence of the coding region cDNA using R. Five of the 13 clones, apparently, contain a complete encoding the amino acid region predicted from this cDNA. Subsequently, one of these clones (clone HGT1) were selected and were analyzed by southern-blotting (using the above probe). As in the case of splitting Hind 111 watched one strip approximately 10 T. p. H., the DNA of this clone was digested and Hind 111 was subjected to agarose gel electrophoresis. The strip 10, etc., ad cut out from the gel, purified and subcloned into the clone vector pUC13, obtaining in the end a clone, we clone pHGT1 was detected, what chromosomal DNA carried by this clone contains the entire coding region for the protein, predicted in example 19, and the nucleotide sequence of the coding region coincides with the predicted sequence of the nucleotide sequence (SEQ 1D 196).

In addition, the area corresponding to codereuse amino acids to exon contained intron 4, and the nucleotide sequence different from the sequence of the complete cDNA clone pHTF1, obtained in example 21 (SEQ 1D 7).

When determining the nucleotide sequences of the remaining 4 clones among 5 chromosomal DNA clones, independently selected by screening using the screening found that 2 of the 4 clones were identical to clone pHGT1, and the other 2 clones were similar to clone GT1, except that they had different nucleotides within the 3'non-coding region, as observed with clone pHTF1 (example 25).

The EcoRI fragment in the thus obtained plasmid clone pHGT1 ligated with expressioni vector PEF18S, receiving expression plasmid pEFHGTE TRO person. After receiving this plasmid DNA (pEFHGTE) and transfection her in COS 1 cells in culture supernatant was detected TPO activity (example 26).

(L)R> The results of examples 18 and 22 revealed that the protein TPO person could exhibit its biological activity even after removing the carboxy-terminal third. Thus, for further analysis of biologically active parts have conducted experiments with deletion derivatives. A number expressing plasmids were prepared using R using DNA plasmid clone RT-231, obtained in example 18 as a matrix and the synthesized oligonucleotides as primers.

Were obtained expression plasmids which contain DNA encoding deletion derivatives TPO person who does not have carboxyl-terminal region of the protein TPO, i.e. deletion derivatives encoding the provisions 1-211, 1-191, 1-171 and 1-163 amino acid sequence. In the preparation of plasmid DNA from each of these clones and transfection her in COS 1 cells TPO activity was detected in each culture supernatant (example 27).

When derivatives were designed with a number of deletions of C-terminal amino acid residues to position 151, and other derivatives with the corresponding deletion of the N-terminal amino acid residues up to the 6th, 7th, and 12th positions, and activity after expression in COS 1 to become netdetective with deletions of amino acid residues N-terminal side to position 7 or deletions of amino acid residues C-terminal side to position 151 (examples 28 and 29).

Derived protein having TPO activity, can be obtained by modifications (deletions, substitutions, insertions, or additions) cDNA that encodes this protein. For this modification you can apply techniques such as PCR, site-directed mutagenesis and chemical synthesis.

(M) the Expression of cDNA TRO person in Cho cells and purification TRO

Was constructed expressing vector, pHTP1 that can Express the cDNA encoding a deduced amino acid sequence TRO person shown in SEQ 1D 6, in animal cells (example 30).

To construct a vector pDEF202-hTPO-P1 used the area cDNA in pHTP1 for his expresii in Cho cell (example 31).

As a result of transfection of this vector in Cho cells and subsequent selection of transfected cells were obtained transfetsirovannyh cells that expressing vector carrying cDNA SRAW man, was integrated into the chromosome Cho cells (example 32).

In example 32 was conducted large-scale cultivation of Cho cell line (Cho 28-30 cells resistant to 25 nm MTX), producing TRO person. Cells were obtained by transferowania expressing TRO person plasmids pDEF202-hTPO-P1 in Cho cells (Prime, TRO person was purified from the culture supernatant in example 55 another method (example 57).

(N) the Expression of cDNA TRO person in H.6.5.3. cells and evidence of his activity

Using district cDNA TRO plasmid pBLTEN obtained in example 30 was constructed expressing vector BMGSneo-hTPO-P1 for use in H.6.5.3. cells (example 33).

After transfection H.6.5.3. cells with this vector were obtained transformants, in which the cDNA encoding human expressing vector was integrated into their chromosomes. When culturing these cells TPO activity was detected in the culture supernatant (example 34).

(A) Expression of large quantities of solid radwaste person in COS 1 cells and purification, measurement mol. mass and biological characteristics SRAW man

Expressing the vector pHTP1 prepared in example 30, was transfusional in COS 1 cells to obtain a large number (in General, approximately 40 l) of culture supernatant containing the expressed product (example 35).

Cleaning TRO was performed from approximately 7 l of cell-free culture supernatant of COS 1 cells obtained in example 35, containing the result of expressing the WGA - column chromatography and kolacny chromatography with reversed phase (example 36).

Partially purified thus TRO from the culture supernatant of COS 1 cells were used for measuring mol. mass and analysis of biological activity (examples 37 and 38).

(B) Expression of TPO person in E. coli

Was constructed in the vector pGEX-2T/h(1-174) for use in the expression of fused protein (referred to as "GST-TPO-(1-174)") glutathione-S-transferase and SRAW man (amino acid residues 1-174) in E. coli. In this case, part of the nucleotide sequence of the cDNA TRO person (approximately half of the zone 5'-side) was replaced by the preferred codons of E. coli (example 39).

GST-TRO(1-174) expressed in E. coli, the resulting cells were destroyed and then solubilizers GST-TPO(1-174) contained in the precipitated fraction. Then, as a result of combining different phases, including testing conditions styling TRO, test conditions purification (affinity column with glutathione, cation exchange column, and so on) and the splitting of the district GST by thrombin, it was possible to perform partial purification of the protein containing the amino acid sequence of the TRO. It was confirmed that this protein detects TPO activity in the test system Krysin what i protein TRO mutated type of person (called "h6T(1-163")), in which the Ser residue in position 1 and the remainder l in position 3 TRO person (amino acid residues 1-163) were respectively replaced by the remainder l and residue Val and Lys residue and the residue Met were respectively added at position -1 and -2. All part of the nucleotide sequence of the cDNA TRO person (corresponding to amino acid residues 1-163) carried this vector was replaced with the preferred E. coli codons (example 42).

h6T(1-163) expressed in E. coli, the resulting cells were literally and then solubilizers h6T(1-163) contained in the precipitated fraction. Experienced conditions for protein folding and partially purified protein containing the amino acid sequence of the TRO. It was confirmed that this protein detects TPO activity in the test system rat CFU-MK (examples 43 and 44).

In addition, was designed vector rsrm/h(1-163) for use in expression in E. coli mutated protein TRO person (called "hM (1-163)"), in which the Lys residue and the residue Met were respectively added to the provisions of 1 and 2. This hMKT(1-163) expressed in E. coli as described in example 43, and the expressed protein was subjected to electrophoresis in SDS-PAGE, transferred to PVDF membrane and then subjected Anatol sequence (example 52).

In addition, was designed vector pCFM536/hMKT(1-332) for expression in E. coli mutated protein TRO person, in which the Lys residue is added at position -1 and the remainder of the C at position -2 TRO person (amino acid position 1-332) (called "hMKT (1-322)"). This h(1-332) expressed inside E. coli as in example 42, and its expression was confirmed by Western blotting using antibodies against peptide TPO person obtained in example 45, as described below (example 66).

(Q) Obtaining antibodies against peptide TPO and cooking columns with antibodies against peptide TPO

Rabbit polyclonal antibodies against TPO peptide was obtained by using synthetic peptides corresponding to the three partial areas amino acid sequence of rat TRO defined in example 10. It was confirmed that these antibodies can recognize molecules SRAW rats and humans. In addition, we synthesized peptides corresponding to 6 partial regions of amino acid sequence TRO person shown in SEQ 1D 6 or SEQ 1D 7), and then applied to the rabbit polyclonal antibodies against TPO peptides. It was shown that the resulting antibodies can learn TRO person (example 45).

(R) Cleaning TRO person, expressed in COS 1 cells, using columns with antibodies against TPO-peptide and mol. mass and biological characteristics TRO

Using the culture supernatant of transfected COS 1 cells (expressing vector rntr) as a starting material was obtained partially purified TRO, which was applied to the column with antibodies against TPO. Because TPO activity was detected in the adsorbed fraction, this fraction was then subjected to column chromatography with reversed phase for protein purification and determination of the mol. mass and biological characteristics (example 47).

(S) confirmation of the activity of partially purified samples TRO person, expressed in COS 1 cells is

TRO-active fraction purified in example 36, namely, a sample of the solid purified to the stage speakers Capcell Pak Cl 300A from the culture supernatant obtained by transfection of COS 1 cells expressing vector pHTP1 about the body (example 48).

Also the crude fraction of the solid obtained by cation exchange column of 33 l of the culture supernatant obtained by transfection of COS 1 cells expressing vector pHTP1, was tested for its biological activity,

to determine whether it is the number of platelets in the body (example 49).

(T) the Expression of the chromosomal DNA TRO person in Cho cells and confirmation activity TRO

Was designed vector pDEF202-ghTPO for use in the expression of chromosome TRO person in Cho cells (example 50).

With the introduction of this vector in Cho cells received cells of the transformant, in which expressive chromosomal DNA vector was integrated into its chromosome. During culturing this cell clone TRO activity was detected in the culture supernatant (example 51).

(U) Partial purification and confirmation of activity of the human TPO mutated type, sexpressions in E. coli

Human TPO mutated type, derived from the coding nucleotide sequence TRO human clone pCF536/h6T(1-163), and expressed in E. coli, were subjected to re-laying using guanidine hydrochloride and glutathione, to determine, does politeceskij TRO mutated type, derived from the coding nucleotide sequence TRO human clone, pCF536/h6T(1-163), and sexpressions in E. coli, were subjected to Repack using N-lauroylsarcosinate of sodium and sulphate of copper, and then cation exchange chromatography, to determine whether the thus purified h6(1-163) the number of platelets in a living body (example 54).

In addition, the laying and clearing of human TPO mutated type, h6T(1-163) was performed with the use of other procedures (examples 60 and 61).

(V) Expression of cDNA TRO person in insect cells and identification of TPO activity

Recombinant virus expression TRO person in insect cells was obtained as described in example 58, and expressed in insect cells Sf 21, after which the solid radwaste activity identified in the culture supernatant (example 59).

(W) the Expression of TPO person (position of amino acids 1-163) in Cho cells and its purification

Was designed vector pDEF202-hTP163 for the expression of protein TRO person (called "hTP163"), having the amino acid sequence 1-163 in amino acid sequence TRO person represented in SEQ 1D 7. Expressing the vector pDEF202-hTP163 was transfusional in Cho cells and Pete. From the supernatant of this culture was purified hTP163 (examples 62-65).

(X) Gets substituted derivatives SRAW man

Was derived (called "N3/TPO"), in which AGD-25 and Glu-231 SRAW man were replaced respectively by Asn and Lys, and derivative (called '09/TRO"), in which His-33 was replaced by Thr.

A plasmid encoding each derivative was transfusional in COS 7 cells, which are then cultivated. The supernatant of this culture was discovered TPO activity.

Were obtained derivatives in which one amino acid h6T(1-163) has been replaced by another amino acid, with the use of the expression system, E. coli TPO activity was detected in each derivative (example 94).

(Y1) Receiving insertion or deletion derivatives SRAW man

The amino acid was introduced in hTPO163 or deletional of him for making insertion or deletion derivatives, then OS7 cells were transfusional plasmids, encoding the obtained derivatives. In the supernatant of these cultures COS 7 cells was detected TPO activity (example 68).

The method of measurement of TPO activity (test system in vitro) used in this invention, described hereinafter referred to as "Standartelecom using peptide fragments TRO person (example 69) and then use Western-blotting (example 70) and in the design of affinity columns with antibodies against TPO (example 71).

(AA) Activity in vivo TPO man

Purified TPO person injected mice with induced thrombocytopenia and changes in the number of platelets observed compared with the control group (example 72) - (example 79). Described pharmaceutical compositions, which can potentially be applied in the treatment of thrombocytopenia (example 80) - (example 89).

(BB) TPO activity as a function of receptor binding

Provided test in which TPO activity determined in units of specific binding with receptor Mr1 (example 90) - (Example 93).

The standard example

A. test for the determination of precursor cells megakaryocytes rats (test system rat CFU-MK) system (liquid culture)

The megakaryocytes include and store serotonin in the cytoplasmic dense granules using an active energy-dependent process (Fedorko, Lab Invest., vol.36, pp.310-320, 1977). This phenomenon can already be seen in the small core of the acetylcholinesterase-positive cells, which are believed to lie between CFU-MK and are distinguished by megakaryocytes (Bricker and Zuckerman, Exp. Hematol., vol. 12, p. 672, 1984). The number of active serotonin is increased in response to increasing the size of megakaryocytes (Schick and Weinstein, J. Lab. Clin. ed. vol. 98, pp.607-6 the Redi bone marrow cells (Schick and Weinstein, J. Lab. Clin. Med. vol. 98, pp. 607-615, 1981). In this test system of highly enriched rat CFU-MK cells (G11b/street 111A+CFU-MK faction, which will be described below) were cultured in the presence of test samples and measure the inclusion of14C-serotonin (14With-hydroxytryptamine-createsurface, 14C-5HT) in megakaryocytes, growing from CFU-MK.

The advantages of this test system is that the indirect influence of the impurity of the cells (for example, education Med-CSF activity impurity cells stimulated by any substance other than the target factor, or the formation of any factor of the impurity cells that are subjected to the combined action of interest to the researcher factor) can be reduced as the percentage FU-MK cells faction G11b/street 111A+CFU-MK is very high (cf follows [test method]), while the number of contaminants (impurities) cells a little. In addition, appropriate culturing conditions can be maintained over a relatively long period, as the total number of cells seeded in one well, very little. Another advantage is that the number of Mature megakaryocytes large, grown from CFU-MK in the presence of active samples during lane is m a qualitative assessment of the presence and degree of activity. The results of the qualitative assessment are in good agreement with the results of quantitative determination, based on the inclusion of 14With serotonin. Thus, the reliability of quantitative determination can be further improved by the simultaneous use of qualitative assessment.

Test method:

Highly enriched rat CFU-MK cells (fraction C-11b/street 111A+CFU-MK) used in this test was prepared by a slightly modifitsirovannom method (Miyazaki et al. (Exp. Hematol., vol. 20, pp. 855-861, 1992).

In short, femur and tibia were removed from Wistr rats (male, 8-12 weeks of age) for the preparation of suspensions of bone marrow cells as described. For the preparation of suspensions of bone marrow cells was used suspenders environment (solution consisting of 13.6 mm trinational salt of citric acid and 11.1 mm glucose, 1 mm adenosine, 1 mm theophylline, 10 mm HEPES (pH 7,25), with 0.5% bovine serum albumin (hereinafter referred to as BSA) (balanced solution Hanks: hereinafter called the "HATCH solution"), which is a slightly modified version of the environment for the Department of megakaryocytes, reported Levine Fedoroko (Levine and Fedoroko, Blood, vol. 50, pp. 713-725, 1977). Thus obtained suspension of bone marrow cells layer on the Prerow is Percoll (Pharmcia) NATS solution and centrifuged at 20oS at 400 x g for 20 minutes. After centrifugation of the extract of cells between layers with density 1,063 and 1,082 g/ml After washing the extracted cells are suspended in culture medium, Dulbecco modified Claims (hereinafter called the cultural environment 1D) containing 10% fetal calf serum (hereinafter referred to as FCS), placed in plastic cups for tissue culture (100 mm) and cultured at 37oC for 1 hour in an incubator with 5% CO2. After incubation, unattached cells isolated and cultured again in plastic cups at 37oC for 1 hour. Unattached cells are suspended in HATCH solution and incubated for 1 hour in bacteriological Petri dishes (100 mm), which were pre-adsorbed mouse monoclonal antibodies against rat platelets Gp11b/111a+, P55 antibody (iyazaki et al., Thromb. Res., vol. 59, pp. 941-953, 1990).

After incubation, unattached cells are removed by thorough washing HATCH solution and the remaining cells adsorbed immobilized P55 antibodies, separated by pipetting and collected. Usually get 3-4105cells from one rat. Thus obtained fraction of cells contains vysokomerie approximately 5-10% FU-MK on the basis of a measurement system analysis of colonies in the presence of saturating concentrations of rat IL-3. Hybridoma, capable of producing the above-mentioned P55 antibody was deposited under Deposit FER BP-4563 National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology. Ministry of International Trade and Industry, February 14, 1994.

Then the cells thus obtained fraction Gp11b/111a+CFU-MK suspended in 1MD culture medium containing 10% FCS, and distributed in portions of 104cells per well 96-well plate to tissue culture. To each well was then added to the standard sample (this will be discussed in details below) or the test sample to bring the final volume to 200 µl per well. Thus prepared the tablet placed in the incubator with CO2and incubated for 4 days at 37oC. On the 4th day of incubation to each well add 0.1 µci (3,7 q)14With serotonin for 3 hours before the end of the cultivation and the end of the incubation continued at 37oC. After 3 hours incubation tablet centrifuged at 1000 rpm for 3 minutes and the resulting supernatant is removed by suction. To each well add 200 ál S containing 0.05% EDTA, and the plate is centrifuged for washing by removal of the resulting supernatant. This stage of the washing cycle is repeated one more time. 200 MK the tablets for approximately 5-10 minutes for complete lysis of cells. Part 150 μl of the thus obtained cell lysates are transferred into commercial solid scintillator in the form of a cap (Cap Ready; Beckman), which is then left overnight at 50oWith in a drying Cabinet for drying lysate. The next day Ready Cap is placed in a glass vial for radioactivity measurement14With using a liquid scintillation counter.

In this case, almost the same results can be obtained in the test include14C-serotonin and when measuring acetylcholinesterase activity in said cell lysate by the method Ishibashi and Burstein (Blood, vol. 67, pp. 1512-1514, 1986).

Standard sample:

First, the blood plasma thrombocytopenic rats were prepared for use in the preparation of standard samples as follows.

Normal male Wistar rats (7 to 8-week) turned in thrombocytopenic by intravenous P55 antibody at a dose of 0.5 mg / animal, twice with an interval of about 24 hours. Approximately 24 hours after the second injection of collected blood from the abdominal aorta under ether anesthesia by injection of 10 ml, which was introduced 1 ml of 3.8% (vol./about.) trinational salt of citric acid as protivosvertyvayushchei at 1200 g for 10 minutes to obtain plasma fractions. Retrieved thus the fraction of blood plasma again centrifuged at 1200g for 10 minutes, and the resulting fraction of blood plasma was collected, avoiding capture consisting of cells, and platelets sediment, etc., and combined (thus obtained blood plasma hereinafter referred to as TRP). Treated calcium TRP (standard sample), the active fraction column WGA-agarose (standard sample W) or active fraction Phenyl Sepharose 6 FF/LS (standard sample R), obtained from TRP in accordance with the procedure of example 1, were dialyzed extensively against a sufficient volume of culture medium 1MDM and used as test standards.

In the process of purification of rat TRO described in example 1, at an early stage was applied standard test With, but then it was replaced with the standard breakdown of W halfway after that and then the standard breakdown of R. the Specific activity of the standard sample was defined as 1 and the relative activity of standard samples W and P was calculated on the basis of this setting. The relative activity of each test sample was determined by comparing the curve depending on the dose response of the standard sample with the curves of the tested samples. The relative activity of the test samples was determined as n, if the BR>
In this test system, bone marrow cells were cultured in semi-solid culture medium in the presence of the test sample and the activity of Meg-CSF was measured by counting the number of colonies of megakaryocytes resulting from proliferation and differentiation of CFU-MK.

Test method

(a) In the case of the use of non-separated bone marrow cells of rats, etc.

1 ml final volume of culture medium 1MDM containing undivided cells rat bone marrow cells obtained in each stage separation Gp11b/111a+CFU-MK test systems or cells Gp11b/111a+CFU-MK faction, and 10% FS, 2 mm glutamine, 1 mm sodium pyruvate, 50 μm 2-mercaptoethanol and 0.3% agar (AGAR NOBLE, made D1FCO), was placed in a plastic Cup for tissue culture (35 mm) and after curing at room temperature were cultured at 37oWith the incubator with CO2. Typically, the number of cells per Cup brought to 2-4105in the case of non-separated bone marrow cells, 2-5104in the case of cells at the stage of density gradient Percoll or depletion by attaching or 0.5-2103in the case of cell fractions Gp11b/111a+CFU-MK. After 6-7 days of cultivation discs of agar was separated from the cups and placed on the glass of the subject with what was homesale to disk in this sequence. Dried thus agar glass was fixed by heating 5 minutes at 50oWith hot plate and soaked for 2-4 hours in Crusader solution acetylcholinesterase according to the method of Jackson (Blood, vol. 42, pp. 413-421, 1973). If a sufficient staining of megakaryocytes were observed, agar glass was washed with water, dried, subjected to post-staining with a solution of Harris hematoxylin for 30 seconds, washed with water and then dried in air. Colonies of megakaryocytes considered 3 or more closely grouped the acetylcholinesterase-positive cells.

(C) In the case of the use unseparated bone marrow cells of the mouse

Test by counting colonies was performed as described in (a) using 2-4105cells for one Cup.

(C) applying cells of human bone marrow or blood cells of the spinal cord of man

You can use cells of human bone marrow or blood cells of the spinal cord of the person as such or in the form of CFU-MK fractions enriched as follows.

First, the liquid bone marrow or blood-spinal cord insist on Lymphoprep (produced by Daiichi Depending Co., Ltd.) and centrifuged, after which the extract obtained in the interphase franciya monoclonal antibodies specific for surface antigens man (CD2, CD11, CD19), can be removed by using connected with Avidya beads. Cells, remove the magnetic beads, are mostly b-cells, T-cells, macrophages and part of granulocytes. The remaining cells stained with FITC-labeled antibodies against CD34 and PE-labeled antibodies against HLA-DR. Then, CD34 and HLA-DR-positive fraction is extracted using a cell sorting device (e.g., ELITE, manufactured COULTER). CFU-MK cells are concentrated in this fraction (hereinafter called XD34+DR+CFU-MK faction). Test by counting colonies of human cells spend the same way as in the case of bone marrow cells of rats, except that used 3-5103cells CD34+DR+CFU-MK faction on one Cup and use a mixture of 12.5% plasma AB man and 12.5% FCS instead of 10% FCS. The cultivation period for the formation of colonies of megakaryocytes equal to 12-14 days. For detection of megakaryocytes conduct immunological staining of megakaryocytes by the method using alkaline phosphatase antibodies against alkaline phosphatase with mouse monoclonal antibodies specific for surface antigen Gp11b/111a of megakaryocytes (for example, Teramura et al. , Exp. Hematol., vol. 16, pp. 843-848, 1988) and is considered astou (M-e test)

M-e cell line megakaryoblasts person, proliferate in response to DM-CSF, IL-3, SCF, IL-2, etc. (Avanzi et al., J. Cell. Physiol., vol. 145, pp.458-464, 1990). Because these cells are also responsible for TRO, they are applicable in the replacement of the test system to the test system rat CFU-MK.

Test method:

M-e cells maintained in the presence of DM-CSF, remove, thoroughly washed and then suspended in culture medium 1MDM containing 10% FCS. The resulting suspension M-e cells is poured into parts of 104cells in wells of 96-well plate to tissue culture and in every hole then add a standard sample or test sample, bringing the final volume to 200 µl per well. Thus obtained tablet was placed in an incubator with 5% CO2and incubated for 3 days at 37oC. After 3 days of cultivation to each well add 1 µci (37 q)3H-thymidine for 4 hours to complete cultivation. After completion of culturing, the cells are collected on filters made of glass fiber by using the harvester (collector) cells to measure3H-radioactivity using a liquid scintillation counter (e.g., Beta Plate, manufactured Pharmacia).

D. the Test system (BA/F3 test), cottora proliferate in response to IL-3, IL-4 and so on ( Palacios et. al., Cell, vol. 41, pp. 727-734). Since its Potsdam F-TE capable of cell proliferation in response not only to IL-3 and IL-4, but also on the TRO, it can be used in the test system, replacing the test with mouse CFU-MK or test cell line megakaryoblasts man (M-e test), as follows. Briefly, BF-TE22 cells growing in the medium 1MDM (1MDM:G1) in the presence of 1 ng/ml murine IL-3, extracted, washed three times 1MDM and suspended in the medium 1MDM containing 10% FCS. Then the cell suspension is poured into wells of 96-well plate to tissue culture at a density of 1104cells per well in each well and then add the standard solution TRO or test sample and bring the final volume to 200 µl per well. The resulting microplate incubated for 2-3 days in an incubator with 5% CO2. On 2-nd and 3-rd day to each well add 1 µci (37 KBq)3H-thymidine and the cultivation continued for another 4 hours. Finally, the cells collected on the filter glass fibre using the harvester cells to measure3H-radioactivity using liquid counter. In this test system, almost all cells, cultured in media without solid radwaste activity, die and, therefore, do not include3H-thymidine. However, cell, cultiv the RO include3H-thymidine. In addition, the results of this test parallel test results M-e and CFU-MK.

The following examples further illustrate this invention.

Example 1-1. Cleaning SRAW rats from plasma thrombocytopenic rats induced by injection of antibodies against platelets. Obtaining blood plasma thrombocytopenic rats induced by antibodies against platelet

R received as a source of purification from about 1000 rats according to the procedure described in "Standard example" above, describe the test system precursor cells of rat megakaryocytes (CFU-MK).

Cleaning SRAW rats from TRP

Source, purification was performed using R 1000 rats, suggesting that the content of the TRO in the TRP should be as 1 to 3 million, and it received a little less than 1 pmol partially purified SRAW rats. Although it is difficult, an attempt was made to analyze its partial amino acid sequences were obtained three partial amino acid sequence, but with low accuracy. These sequences were identical or were close to the amino acid sequence of the inhibitor of serine proteases (SP1), produceren the SHL, or the sequences derived from the sequences of impurity proteins other than TPO. Using these uncertain sequences were cloned gene SRW from cDNA library rat, but was unable to get potential coding TRO genes. After that conducted intensive research based on the assumption that this failure was due to the low degree of purity and the insufficient number of analyzed samples. To obtain the final purified samples required for amino acid analysis, purification was performed in accordance with the process described in the following example 1-2. Unexpectedly, the results of example 1-2 showed that the content of the TRO in R or XRP (described below) was so extremely low as 1:100000000 - 1 bln. total plasma proteins, so clear TRO was extremely difficult.

Example 1-2. Cleaning SRAW rats from plasma thrombocytopenic rats, induced by irradiation of the whole body x-rays or-rays [plasma thrombocytopenic rats, induced by irradiation of the whole body x-ray or rays]

Normal male Wister rats (7-8 weeks) were transformed in thrombocytopenic irradiation of the whole body x-ray is azmy blood (hereinafter called XRP) was prepared in the same way, as described above for the preparation of TRP.

In this case, XRP (approximately 8 l) was obtained from 1100 rats and used as source material (source) for cleanup.

Cleaning SRAW rats from R

The sample of blood plasma from 1100 rats was too great for one-time processing because of its content of total protein was achieved 493000 mg. Therefore, a sample of blood plasma was divided into 11 episodes, each corresponded to about 100 rats, in the following stages of treatment (1)-(4). In further stages of treatment (5)-(7) the sample was divided into 6 groups. In and after the cleanup phase (8) was applied to the sample, roughly purified from total blood plasma is approximately 1100 rats. Further described is a typical example of each stage of treatment in the case series (XW9) and group (HV).

All steps of purification TPO activity was measured using the test system rat CFU-MK, described in "Standard example". Unless otherwise indicated, all stages of the purification were performed at 4oWith, except chromatography with reversed-phase and gel filtration on Superdex 75 pg in the presence of surfactants, which were carried out at room temperature. The protein determination was performed in the test binding of the dye Kumasi system (reagent, manufactured PIERCE, tx2">

The purification scheme is shown in table 1.

(1) Treatment with calcium chloride, centrifugation and treatment with protease inhibitor plasma of rats

In the case of series XW9

XRP (742 ml, a protein concentration of 54.8 mg/ml, total protein 40 686 mg), equivalent to about 100 rats, stored at -80oC, thawed and poured into polypropylene centrifuge tubes (manufactured Nalgene). Powder of calcium chloride was added in each tube to a final concentration of 100 mm. After incubation overnight at 4oWith the resulting mixture was centrifuged at 8000 rpm for 60 minutes. To the obtained TRO-active supernatant (742 ml, protein concentration 54.9 mg/ml, total protein 40 740 mg) was added protease inhibitor p-APMSF ((p-aminodiphenyl)methanesulfonate, hydrochloride, manufactured WAko Pure Chemical Industries, Ltd., cat. 010-10393) to a final concentration of 1 mm. Thus obtained sample was used in further stages of buffer exchange on a column of Sephadex G-25.

In this way were processed by a calcium chloride/ p-APMSF all R derived from 1100 rats (total 8184 ml, total protein 493000 mg) divided into 11 episodes, each of which corresponded to about 100 rats, and treatment of these series is"ptx2">

(2) Sephadex G-25 (change buffer)

In the case of series XW9

The supernatant obtained after calcium treatment stage (1) (742 ml, protein concentration 54.9 mg/ml, total protein 40 740 mg), was applied at a rate of flow 40-70 ml/min at a column of Sephadex G-25 (produced Pharmacia Biotech, cat. 17-0033-03, the diameter of 11.3 cm, height of Packed column 47 cm), equilibrated in advance 20 mm Tris-HC1, pH 8. The elution was performed with the same buffer. Portion 1300 ml of the eluate before elution of protein was thrown out. When UV absorption was detected in the eluates, fractions were collected until the conductivity reached 500 S/cm, thus removing TRO-active protein fraction containing the now 20 mm Tris-HC1, pH 8 (1377 ml, protein concentration 27,56 mg/ml, total protein 37882 mg, protein yield 93%). The relative activity of TPO activity in this fraction was 2.3.

As a result of processing on a column of Sephadex G-25 all series of samples was obtained in whole 21117 ml TRO-active fractions (total protein 480300 mg, the average relative activity 1 total activity 864600).

(3) the Q-Sepharose FF (strong anion exchange chromatography)

In the case of series XW9

TRO-active fraction obtained in stage (2) processing Sephadex G-25 (1377 ml, a protein concentration of 27.5 mg/ml, total protein 37841 mg, the relative activity 2,3), who believed 20 mm Tris-HC1 (pH 8) and the fraction F1, passing through the column were collected (3949 ml, a protein concentration of 0.98 mg/ml, total protein 3870 ml, the relative activity 0).

Then the buffer was replaced with 20 mm Tris-model HC1 (pH 8) containing 175 mm such as NaCl for elution TRO-active fraction F2 (4375 ml, protein concentration are 5.36 mg/ml).

Finally fraction F3 (1221 ml, a protein concentration of 3.9 mg/ml, total protein 4783 mg, the relative activity of 3.8) was suirable 20 mm Tris-model HC1 (pH 8), containing 1000 mm NaCl. Total protein in TRO-active fraction F2 was 23440 mg and the yield of protein F2 at this stage was of 61.9%. In addition, the relative activity SRAW increased to 6.8.

As a result of applying all series SRW-active fractions after Sephadex G-25 to Q-Sepharose FF received generally 35842 ml TRO-active fraction F2 (total protein 314384 mg, the average relative activity 9 total activity 2704000).

(4) Agglutinin wheat germ (WGA)-Agarose (pectin affinity chromatography)

In the case of series XW9

TRO-active fraction F2, obtained in stage (3) using Q-Sepharose FF, divided into 3 parts and put on WGA-Agarose (produced Honen Corp. cat. 800273, diameter 5 cm, height 22.5 cm) at a flow rate of 5 ml/min and was suirable isotonic phosphate buffer of Dulbecco (DPBS). fraction F1 passing through the column were collected (9336 ml, the end of the M buffer, containing sodium phosphate (pH of 7.2) containing 0.2 M N-acetyl-D-glucosamine (GlcNAc, manufactured by Nacalai tesque cat. 005-20), 150 mm NaCl and 0.02% sodium azide, and the resulting eluate were combined and concentrated using ultrafiltration element (Omega Ultrasette, nominal clipping mol. weight 8000, manufactured Filtron), resulting in the TRO-active fraction F2 (2993 ml, protein concentration 0,376 mg/ml).

Total protein in TRO-active fraction was 1125 mg and the yield of protein F2 this stage was 4.8 percent. In addition, the relative activity SRAW was increased to 101. Thus obtained fraction F2 was stored at -80oC.

As a result of applying all series SRW-active fractions Q-Sepharose FF on WGA-Agarose received generally 33094 ml WGA-Agarose TRO-active fraction F2 (15030 total protein mg, the average relative activity 132 total activity 1987000).

(5) TSK - gel AF-BLUE 650 MN (affinity chromatography with triazine dye)

In the case of a group HW

WGA-Agarose, absorptive TRO-active fraction series XW9, and WGA-Agarose, absorptive TRO-active fraction F2 series XW9 obtained in stage (4) corresponding to the source 215 rats, were United in a group HV (5947 ml, protein concentration 0,388 mg/ml, total protein 2319 mg, the relative activity is 150).

5974 the th concentration of NaCl 0,822 M, and the resulting solution was applied at a flow rate of 7 ml/min, TSK - gel AF-BLUE 650 MN-column (TOSOH CORP. cat. 08705, diameter 5 cm, height of the column 23 cm), which was pre-equilibrated to 20 mm sodium phosphate buffer (pH of 7.2) containing 1 M NaCl.

After applying the protein was suirable 20 mm sodium phosphate buffer (pH of 7.2) containing 1 M l at flow rate 10 ml/min Received eluate were combined and concentrated using ultrafiltration element (Omega Ultrasette, nominal clipping mol. mass 8000), receiving passing fraction F1 (543 ml, a protein concentration of 2.05 mg/ml), total protein 1112 mg, the relative activity 31).

Then eluting buffer was replaced with 2 M NaSCN to get elyuirovaniya TSK-gel AF-BLUE 650 MN absorbed TRO-active fraction F2 (1427 ml, protein concentration 0,447 mg/ml).

Total protein in TRO-active fraction F2 was 638 mg, and the yield of protein F2 at this stage was 27.5 per cent. In addition, the relative activity SRAW was increased to 1500.

As a result of applying all groups WGA-Agarose absorbed TRO-active F2 fractions on TSK-gel AF-BLUE 650 MN received generally 10655 ml TRO-active fraction F2 (total protein 4236 mg, the average relative activity 905 total activity 3834000).

(6) Phenyl Sepharose 6 FF/1424 ml, the protein concentration 0,447 mg/ml, total protein 638 mg, the relative activity of 1500) was mixed with 1.5 mole of a powder of ammonium sulfate in 1000 ml (282,2 g in General), obtaining a solution with the final concentration of ammonium sulfate of 1.35 M

The resulting solution was applied at a flow rate of 7 ml/min on a Phenyl Sepharose 6 FF (Low Sub) column (Pharmacia Biotech, cat. 17-0965-05, diameter 5 cm, height of column 10 cm) that was equilibrated in advance with 50 mm sodium phosphate buffer (pH of 7.2) containing 1.5 M ammonium sulfate. After applying the elution was performed using 36 mm sodium phosphate buffer containing 0.8 M ammonium sulfate, at a flow rate of 10 ml/min Received eluate (approximately 3160 ml) were combined and concentrated using ultrafiltration element (Omega Ultrasette, nominal clipping mol. mass 8000), receiving a fraction F1 (485 ml, protein concentration 0,194 mg/ml, total protein was 94.2 mg, the relative activity 0).

Then eluting buffer was replaced with 20 mm sodium phosphate buffer (pH 7,2) to obtain elyuirovaniya TRO-active fraction F2 (approximately 2500 ml). Elyuirovaniya fraction was concentrated using ultrafiltration element (Omega Ultrasette) and took a sample for analysis. At this stage the concentration of protein and total protein TRO-akt activity TRO was 1230.

As a result of applying all groups TRO-active F2 fractions of TSK-gel AF-BLUE 650 MN on hnl Sepharose 6 FF/LS has received a total of 1966 ml fractions F2 (total protein 2762 mg, the average relative activity 847 total activity 2339000).

(7) Sephacryl S-200 HR (gel chromatography)

In the case of a group HV (see Fig.1)

TRO-active fraction F2 from hnl Sepharose 6 FF/LS (217 ml, a protein concentration of 1.45 mg/ml, total protein 315 mg, the relative activity 1230) was mixed with 144,8 ml of 5 M NaCl, getting 362 ml of a solution with a final concentration of 2 M NaCl, and the resulting solution was concentrated to approximately 50 ml using ultrafiltration devices with UM membrane (76 mm in diameter, Amicon Corp.).

To this solution was added an equal volume (50 ml), 8 M urea, getting about 100 ml of a solution containing 1 M NaCl and 4 M urea in the form of final concentration. It was concentrated to about 80 ml, took a sample 88,78 ml and was applied to a column of Sephacryl S-200 HR (Pharmacia Biotech, cat. 17-0584-01, 7.5 cm in diameter and 100 cm in height).

Thereafter, elution was performed with PBS flow rate 3 ml/min, and eluate after the empty volume of 1200 ml were collected portions 45 60 ml polypropylene tubes. The test was performed after every two test tubes and other tubes were stored at -85othe Tobit analyses faction group HV were grouped as follows.

(F1) the number of tubes 1-15 (fraction near empty volume, mol. mass 94000 or more)

(F2) the number of tubes 16-26 (mol. mass 94000-33000)

(F3) non tubes 27-44 (mol. weight 33000-3000)

(F4) non tubes 45-55 mol. weight of 3000 or less)

Thus all groups F2 fractions (TRO-active), obtained using Phenyl Sepharose 6 FF/LS, separately applied on a Sephacryl S-200 HR, analyzed and stored at -85oC. After this treatment, all groups and directly before the subsequent operation chromatography with reversed phase YMC-Pack PROTEIN-RP) these stored samples were thawed and concentrated using ultrafiltration devices with MIND 3 membrane (76 mm in diameter, Amicon Corp.), getting these two samples. This concentrated sample TRO-active fractions F2, obtained after Sephacryl S-200 HR, hereinafter referred to as "break the F2 molecular TRO, and F3 - "break the F3 low-molecular TRO".

For convenience, the sample F2 high-molecular solid and sample F3 low-molecular TRO are combined fractions from the different areas of elution gel chromatography, and the terms "high molecular weight" and "low molecular weight", therefore, cannot signify their true mol. mass. (see tab.I)

Sample F3 low-molecular TRO and sample F3 is kularnava TRO described in the following stages (8)-(11).

(8) YMC-Pack PROTEIN-RP (chromatography with reversed phase)

Sample F3 low-molecular TRO (total protein to 50.3 mg, protein concentration 0,184 mg/ml, the relative activity of 20,000 total activity 1007000, total 274 ml) obtained in stage (7), was mixed with solvent A (0.025% of triperoxonane acid (TFA) and solvent B (1-propanol containing 0.025% TRA), obtaining a solution having a total volume 508,63 ml and a final concentration of propanol, TFA and protein about 20% 0,012% and 0,0989 mg/ml, respectively. Insoluble material formed during the preparation of this solution was separated by centrifugation and the resulting supernatant was divided into two parts, 254,3 ml of 25.2 mg protein) and was applied at a flow rate of 2 ml/min at a column Packed YMC-Pack PROTEIN-RP (EMS, cat. A-RR-33-03-15, 3 cm diameter, 7.5 cm, the height of the column, which was pre-equilibrated 30% C. the Precipitate formed during the centrifugation, was dissolved in 20 ml of 20 mm sodium acetate (pH 5.5) containing 5 mm CHAPS (sulfonate 3-[3-cholamidopropyl)dimethylammonio] -1-propane, manufactured Dojindo Laboratories, cat. 75612-03-3) and also were applied to this column.

After application of the sample to approximately 50 ml of solvent (solvent A: b= 3: 1) was passed through the column to obtain p is 45%) and eluate collected in 36 polypropylene test tubes of 10 ml in each tube. This process was repeated for the remaining samples using the same tubes for collection of fractions, thus receiving in total, 36 test tubes, each containing 20 ml of the eluate. Passing through the column fraction was immediately concentrated to 20 ml using ultrafiltration devices with MIND 3 membrane (76 ml in diameter, manufactured by Amicon Corp.).

0.1 ml passing through the column fractions and each of 20 ml fractions of the test tubes with the numbers 1-36 was mixed with 20 µl of 5% And was dried by evaporation, dissolved in 0.25 ml of the test environment 1MDM and then analyzed to identify TRO-active fractions. In the TPO activity was detected in fractions test tubes with numbers 17-27 (range of concentrations of propanol 36,0-43,0%), which were combined and used as YMC-Pack PROTEIN-RP TRO-active fraction F2 obtained from the sample F3 low-molecular TRO. This fraction was stored at -85oTo its use in the subsequent stage using a YMC-Pack CN-AP.

YMC-Pack PROTEIN-RP TRO-active fraction F2 obtained from the sample F3 low-molecular TRO

Total volume of 220 ml

The concentration of protein - 0,0130 mg/ml

Total protein - to 2.85 mg

Relative activity - 130000

Total activity - 371000

(9) YMC-Pack CN-AP (chromatogram TRO (total protein and 2.79 mg, the protein concentration 0,0130 mg/ml, the relative activity 130000 total activity 36300) obtained from stage (8), was mixed with 0.6 ml of 50% glycerol and concentrated to 1.8 ml of This concentrate is then brought up to a volume of 5 ml containing 20% or less propanol and approximately 6% of glycerin.

The concentrate was divided into 5 parts for use in the next column operations (0,555 mg protein and 1 ml volume for each operation). Each of the divided thus sampling was applied at flow rate of 0.6 ml/min at a column Packed YMC-Pack CN-AP (EMS, cat. AR-513, 6 mm in diameter and 250 mm height), which was pre-equilibrated with 15%, with 0.1% TFA as solvent a and 0.05% of TFA containing 1-propanol as solvent Century After the application of the propanol concentration was increased from 15% to 25% In and spent 65 minutes linear gradient from 25% to 50% Century After the final (5th) operation 1 ml of a solution having the same composition, except for protein, was applied on the column and treated in the same way, to extract SRW activity retained by the column. Because it used the same polypropylene test tubes to collect the eluates in 6 operations, has received a total of 44 tubes, each of which consisted of 7.2 ml of the eluate.


test tubes with numbers 28-33 (37,0-42,0% concentration range propanol), which were combined and used as YMC-Pack CN-AP basic TRO-active fraction F obtained from the sample F3 low-molecular TRO.

YMC-Pack CN-AP SRW-active fraction F obtained from the sample F3 low-molecular TRO

Total - 43,20 ml

The concentration of protein - 0,00863 mg/ml

Total protein - 0,373 mg

Relative activity - 800000

Total activity - 298400

(10) Capcell Pak C1 300 (final chromatography with reversed phase)

43,12 ml of 43,20 ml TRO-active fractions F obtained from the sample F3 low-molecular TRO (total protein 0,372 mg, protein concentration 0,00863 mg/ml, the relative activity 800000 total activity 297500) obtained in stage (9), was mixed with 0.2 ml 50% glycerol and concentrated to obtain 0.1 ml glycerol solution.

This solution was mixed with 2 ml of solvent A (0.1% OF F): solvent (1-propanol containing 0.05% F) = 85:15 (15%), to get to 2.1 ml of sample containing approximately 14% of propanol, approximately 4.8% of glycerol and 0,177 m is Ltd., cat. C1 TYPE:SG300A; 4.6 mm in diameter and 250 mm height), which was pre-equilibrated with 15%, and showed a 65 minute linear gradient from 27% to 38% when the speed of the current of 0.4 ml/min

Eluate collected in 72 polypropylene tube (0.6 ml in each tube.

3 μl of each of the thus obtained fractions (1/200 part of each fraction) was mixed with 20 μl of 5% BSA, the medium was replaced to 225 μl of the test environment 1MDM and analyzed dissolved in 75 times a fraction.

1 ál of each fraction (1/600 fraction) were taken for use in electrophoresis, dried by evaporation and treated with 95oWith 5 minutes 10 ál of non-sample buffer for SDS-PAGE. The thus treated sample was subjected to electrophoresis SDS-PAGE using 15-25% SDS-polyacrylamide preformed gel (manufactured Daiichi Pure Chemicals Co., Ltd) and then stained with silver from a set of 2D-Silver Stain-11 "DIII" (manufactured Daiichi Riga Chemicals Co., Ltd., cat. 167997, referred to as set for silver staining). As markers mol. mass used /DAIICHI/-111 low molecular weight markers (manufactured Daiichi Pure Chemicals Co., Ltd., cat. 181061, hereafter referred to as "DPC111").

The result of this analysis, significant TPO activity was detected in fractions preconcentrate propanol to 30.5-32.0 per cent) were combined and used as the main TRO-active fractions F. These results are shown in Fig.2.

When the protein content set of the chromatogram was compared with these analysis results, it was found that the thus obtained fraction had a total protein 39,6 mcg, the protein concentration of 9.4 mg/ml, the relative activity 4890000 and General activity 193600. The study of the distributions of SDS-PAGE SRW-active fractions with numbers 36-42 revealed the presence of bands, the density of staining which correlates with the strength of activity. In addition, the apparent mol. the weight of this unrestored band was 17000-19000 when compared with mol. masses of standard proteins with known mole. masses on the same gel were recovered, indicating that this band is in a strong degree candidate TRO.

(11) Extraction TRO-active protein from gel electrophoresis (15% SDS-PAGE)

Example analysis TRO-active fractions F

From 4200 µl TRO-active fractions F obtained from the sample F3 low-molecular TRO (total protein was 39.6 µg protein concentration of 9.4 mg/ml, the relative activity 4890000 total activity 193600) obtained in stage (10), and 5.5 μl (1/764 faction) for use in the extraction of active protein and 2.5 μl (1/1680 faction) for use in staining silver p is the buffer for samples for SDS-PAGE at 37oC for 1 hour and then left to stand until 18 hours at room temperature, which contributed to the reaction SDS.

Pre-painted marker low range (Bio-Rad Laboratories, Inc., 161-0305) and the aforementioned DPC111 used as markers mol. mass. Applying microplastic gels, electrophoresis on 15% SDS-PAG these samples was carried out at 4oWith the conventional method (Laemmli, Nature, vol. 227, pp.680-685, 1970). After completion of the electrophoresis gel parts intended for silver staining, immediately cut with a scalpel from the gel, placed in fixative and then stained using the above kit silver staining.

Apart from this the entire range of mole. mass of gel that is designed to detect the activity, cut with a scalpel 34 slice and each slice having a width of 1.5-2.5 mm, was defeated by a slightly modified method Kobayashi (Kobayashi, M. , Seikagaku (Biochemistry), vol.59, N9, 1987, published by the Japanese Biochemical Society). Each gel slice, divided thus into small pieces, mixed with 0.3 ml of buffer for extraction (20 mm Tris - Hcl, pH 8, 500 mm NaCl, 0,05% BSA) and rocked for 6 hours at 4oWith to enhance extraction.

To this suspension was added nosily in Ultra Free C3GV 0.22 μm filter unit (Millipore Corp., UFC3 OGVOS) and centrifuged at 1000 g (4000 rpm) for 15 minutes to remove precipitated SDS and extraction of the resulting supernatant. The filtrate is used in Ultra Free C3-LGC ultrafiltration device (nominal clipping mol. weight of 10,000, Millipore Corp., UFC3 LGC 00) and centrifuged at 3000 g (7000 rpm). When the amount of concentrate solution reached approximately 50 μl, was added 300 μl of 20 mm sodium phosphate buffer (pH 7,2) and again subjected to ultrafiltration.

Ultrafiltration using 300 μl of 20 mm sodium phosphate buffer was repeated twice to remove any remaining SDS. Following that stage was repeated for the exchange of the culture medium for preparation of samples (300 µl), which is then sterilized and subjected to measurement of TPO activity.

Three protein bands were clearly detected with silver staining, which showed average mol. weight approximately 17000-19000, 11000 14000 and, according DPC111 markers mol. mass.

In the previous stage (10) strip having a correlation between the strength of the activity and density of staining and with an average mol. weight approximately 17000-19000 observed in the electrophoresis TRO-active fractions Capcell Pak column C1. In the experiment this stage (11) TPO activity was defined above results, it was confirmed that that TRO-active protein was purified active fractions Capcell Pak C1 300A column to the detected level on the gel for electrophoresis. On the basis of the density of silver staining of this band, received on 15% SDS-PAGE, it was determined that the number of candidate protein TRO (average mol. weight approximately 17000-19000) in General TRO-active fraction is approximately 1.7 µg.

Wipe samples F2 macromolecular TRO described in the following stages (12)-(15).

(12) YMC-Pack PROTEIN-RP (chromatography with reversed phase)

Sample F2 macromolecular TRO (total protein 257 mg, protein concentration 0,894 mg/ml, the relative activity 7840 total activity 2015000, total 287 ml) obtained in stage (7), was mixed with 95,8 ml (1/3 of the sample volume) solvent (1-propanol containing 0.025% F) to obtain a solution having a total volume 383 ml and a final concentration of propanol, F and protein approximately 25%, 0,006% and 0,671 mg/ml, respectively. A solution of 0.025% F used for solvent A. the Insoluble material formed during cooking of the applied samples were separated by centrifugation and the resulting supernatant was divided into six parts of 62.3 ml (42,8 ml protein) was applied at a flow rate of 2 ml/min at a column of upakovan Century The precipitate obtained by centrifugation, was dissolved in 10 ml of 20 mm sodium acetate (pH 5.5) containing 5 mm CHAPS, and also were applied to this column.

After application of the sample to approximately 50 ml of solvent (solvent A: b= 3:1) was passed through the column to obtain passing through the column fractions. Then began manifesting program (120 min linear gradient from 30% to 45%), and eluate collected in 24 polypropylene tubes 15 ml in each tube. This process was repeated for 6 divided samples using the same tubes for collection of fractions, and thus, in General, 24 tubes, each of which contained 90 ml of the eluate. Passing through the column fraction and the fraction tubes 1 were combined and then concentrated to 90 ml using ultrafiltration devices with UM membrane (76 mm in diameter, Amicon Corp.).

0.3 ml passing through the column fractions plus the fraction of the tube 1 and each of the fractions tubes 2 - 24 was mixed with 10 μl of 5% BSA, dried by evaporation, dissolved in a 0.30 ml of the test culture medium IMDM and then analyzed to identify TRO-active fractions. In the TPO activity was detected in fractions test tubes with the numbers 10-15 (range of concentrations of propanol 34,0-39,5 warnowo TRO. This fraction was stored at -85oTo use in the subsequent phase YMC-Pack CN-AP.

YMC-Pack PROTEIN-RP TRO-active fraction F2 obtained from the sample F2 macromolecular TRO

Total volume of 540 ml

The concentration of protein - 0,021 mg/ml

Total protein - 11.4 mg

Relative activity is 227000

Total activity - 2588000

(13) Superdex 75 rd (gel chromatography in the presence of CHAPS)

538,2 ml originating from the sample F2 macromolecular TRO YMC-Pack PROTEIN-RP TRO-active fraction F2 (total protein of 11.3 mg; protein concentration 0,021 mg/ml; relative activity 227000 total activity 2565000) obtained in stage (12), was mixed with 0.6 ml of 50% glycerol and concentrated by evaporation. Then was added 18 ml 20 MCHAPS, followed by mixing and incubation for 41 hours at 4oC. After this first sample was applied to a column Packed iload 26/60 Superdex 75 EP (produced Pharmacia Biotech, cat. 17-1070-01, 2.6 cm in diameter and 60 cm high) and were suirable DPBS containing 5 mm CHAPS at flow rate 1 ml/min Part 4 ml of each sample (protein concentration 0,466 mg/ml protein to 1.86 mg) was applied to this column.

In this case, the operation of the column Superdex 75 was repeated 6 times by dividing the entire UMC-Pack PROTEIN-RP TRO-active fractions into 6 parts. Eluate each operation column operations.

0.1 ml of each of the thus obtained fractions were mixed with 10 μl of 5% BSA, dried by evaporation, dissolved in 0.25 ml of IMDM and then analyzed to identify TRO-active fractions. In the TPO activity was detected in fractions test tubes with numbers 13-31 (mol range. masses from 78,000 to 3000), which were combined and used as obtained from the sample F2 macromolecular TRO Superdex 74 rd TRO-active fraction F2.

Obtained from the sample F2 macromolecular TRO Superdex 75 rd TRO-active fraction F2

Total volume of 540 ml

The concentration of protein - 0,00216 mg/ml

Total protein - 1,17 mg

Relative activity - 1750000

Total activity - 2041000

(14) YMC-Pack CN-AP (chromatography with reversed phase)

513,2 ml 540 ml obtained from the sample F2 macromolecular TRO Superdex 75 rd TRO-active fraction F2 (mol. weight 78000-3000) (total protein 1.11 mg, protein concentration 0,00216 mg/ml, the relative activity 1750000 total activity 1943000) obtained in stage (13), was mixed with 1/10 volume of solvent (1-propanol containing 0.05% F) and was applied at flow rate of 0.6 ml/min at a column Packed YMC-Pack CN-AP (izgotovliaemye UMC, cat. AR-513, 6 mm in diameter and 250 mm height), which was pre-equilibrated with 15%, C % to 25% In and spent 65 minutes linear gradient from 25% to 50% Century

Before chromatography column described 1/20 part of the total deposited samples were tested to confirm proper extraction activity. After that the remaining 19/20 samples were divided into 2 samples for holding a column operation twice. In other words, the operation column was repeated three times. Since the same tubes used to collect the eluates in 3 operations, only received 44 tubes, each of which contained 3.6 ml of the eluate.

5 ml of each of the thus obtained fractions (1/720 part of each fraction) was mixed with 0.25 ml of IMDM and then analyzed to identify TRO-active fractions. In the TRO activity (strong enough) was detected in the fractions of the test tubes with the numbers 24-30 (the range of concentrations of propanol 36,0-42,0), which were combined and used as received from the sample F2 macromolecular TRO YMC-Pack CN-AP basic TRO-active fraction F.

Obtained from the sample F2 macromolecular TRO YMC-Pack CN-AP SRW-active fraction F

Total - 25,20 ml

The concentration of protein - 0,0246 mg/ml

Total protein - 0,620 mg

Relative activity - 700000

Total activity - 434000

(15) Capcell cancer C1 300A (final chromatography with reversed phase)
06 mg, the protein concentration 0,0246 mg/ml, the relative activity 700000 total activity 424000) obtained in stage (14), was mixed with 0.4 ml of 50% glycerol and concentrated by evaporation.

In this way there were obtained 2 ml concentrated sample with the concentration of propanol several %, the concentration of glycerol (10%) and protein concentration 0,303 mg/ml was applied to a column Packed Capcell Pak C1 300A (izgotovliaemye Shiseido Co., Ltd., cat. C1 TYPE:SG300A, 4.6 mm in diameter and 250 mm height), which was pre-equilibrated with 15%, using solvent A (0.1% OF F) and solvent B (1-propanol containing 0.05% F) and showed a 65 minute linear gradient from 27% to 38% when the flow rate 0.4 ml/min Eluate collected in 72 polypropylene tube (0.6 µl in each tube.

0.75 μl of each of the thus obtained fractions (1/800 part of each fraction) was mixed with 20 μl of 5% BSA, the medium was replaced to 225 μl of IMDM and analyzed diluted so 300 times the fraction.

2 μl of each fraction (1/300 faction) preparing samples for electrophoresis, dried by evaporation and treated with 95oC for 5 minutes, 10 μl of non buffer for samples for SDS-PAGE. The thus treated sample was subjected to SDS-PAGE with a note with application kit silver staining. The above DPCIII used as markers mol. mass.

As a result of this analysis a significant TPO activity was detected in fractions test tubes with numbers 33-39 (range of concentrations of propanol to 29.5-31.5 per cent). Faction tubes 34-39 (range of concentrations of propanol and 30.0-31.5 per cent) were combined and used as the main TRO-active fraction A. the Use of SDS-PAGE distribution main TRO-active fraction revealed the presence of bands, the density of staining which correlates with the strength of activity within secondary mol. mass 17000-19000 when non conditions, which was similar to the band if obtained from samples F3 low-molecular SRW SRW-active fractions, as described in stage (10).

Example 2. Analysis of partial amino acid sequences of purified SRAW rats

Amino acid sequence analysis of the candidate protein SRAW rats in Capcell Pak C1 300A TRO-active fractions F obtained in the stage of purification (10) of example 1 was performed according to the procedure Iwamatsu (Iwamatsu et al., "Shin Kiso Seikagaku Jikken-ho (New Basic Biochemical Experiments), vol.4, pp. 33-84, pub. Maruzen; Iwamatsu, A, Seikagaku (Biochemistry): vol. 63, No 2, pp.139-143, 1991; Iwamatsu, A., Electrophoresis, vol. 13, pp.142-147, 1992): I.e., the sample was subjected to SDS-PAGE and transferred electric sposobnostey thus protein was digested to peptide fragments in a systematic and stepwise hydrolysis using restrictively enzymes in situ three proteases and the resulting peptide fragments at each stage of cleavage was separated and purified by chromatography with reversed phase and analyzed for their amino acid sequences using a highly sensitive method for determining the amino acids. Further, this process is described in detail.

Example of analysis of protein-candidate TRO received from the sample F3 low-molecular TRO Capcell Pak C1 300A faction TRO F

(1) the Concentration Capcell Pak C1 300A faction TRO F

From 4200 µl obtained from the sample F3 low-molecular SRW SRW-active fractions F with speakers Capcell Pak C1 300A (number of tubes 36-42) obtained in stage (10), (total protein was 39.6 µg protein concentration of 9.4 mg/ml, the relative activity 4890000 total activity 193600), 4151 ál (98.8% of all fractions) was applied in the analysis of sequences of amino acids. Total protein, calculated from the chromatogram, was 39,1 mcg, with 1.6 µg was estimated protein TRO, painted silver after SDS-PAGE and having the average mol. weight approximately 17000-19000.

This sample was mixed with glycerin and concentrated by evaporation, receiving 5 ál of glycerol solution. To it was added a non buffer for SDS-RADA and 1 M Tris-model HC1 (pH 8) to adjust its pH, receiving approximately 25 μl of sample containing 200 mm Tris-model HC1 (pH 8), 50 mm Tris-model HC1 (pH 6.8), 1,1% SDS, 2 mm EDTA, 0.02% bromophenol blue and 30 g of glycerin.

Thus obtained sample was kept at room temperature for 14 hours and then treated at 60 Prepared microplastic gels (4,0% acrylamide concentrating gel and 15% acrylamide separating gel) and SDS-PAGE was carried out at room temperature for 2 hours at a constant current of 12.5 mA and then to 17.5 mA. Pre-painted Low Range Marker (Bio-Rad, 161-0305) and DOCIII used as markers mol. mass. Immediately after electrophoresis of the obtained protein molecules were transferred to PVDF membranes (see next stage).

Part of this sample was also used in another electrophoresis using 15-25% preformed polyacrylamide gel (Gel Multi 15/25, made Daiichi Pure Chemicals Co., Ltd., 211072) when non conditions or after recovery, samples dithiothreitol (DTT). When the obtained gel was stained using the above kit silver staining, it was confirmed that the mol. the mass of the protein bands of the proposed TRO was approximately 19000 under reducing conditions and cleanliness of the proposed TRO protein with a column Capcell Pak C1 300A was only a few percent. In addition, since the mobility of this band was wariyapola when and non reducing conditions, an assumption was made that the protein candidate contains at least one disulfide bond.

ProBlott, Applied Biosystems; cat. 400994) was performed for 1 hour at a constant current of 160 mA (11-17) using semi-dry device for migration (Model KS-8460, manufactured Marysol). A solution consisting of 0.3 M Tris and 20% methanol (pH 10) was used as the anolyte, the solution consisting of 25 mm Tris and 20% methanol (pH 10,4), was used as a solution for membrane and a solution consisting of 25 mm Tris, 40 mm aminocaproic acid and 20% methanol (pH 10,4) as Catolica.

When coloration is transferred thus membrane dye solution of Roseae S (0,1) g Ponceau S and 1 ml acetic acid in 100 ml of water) found many of the bands and it was confirmed that the proposed TRO protein was saying. the weight of 19000. This band was cut out for use in subsequent stages of education peptide fragments.

(4) Formation of peptide fragments, peptide mapping and analysis of amino acid sequences

To perform a systematic (directed) fragmentation of the proposed TRO protein transferred to the membrane and S-alkylated after recovery on a PVDF membrane, manual limited enzymatic hydrolysis was carried out using the following three proteases.

The first cleavage: l the tion: endoproteinase ASp-N (manufactured Boeringer-Mannheim Corp., cat. 1054 589)

The third cleavage: trypsin-TRNC (manufactured Worthington Biochemical cat. 3740)

Peptide fragments resulting from each enzymatic degradation, was applied on the column, Wakosil-11 S C18 reversed-phase (Wako Pure Chemical Industries, Ltd., 2.0 mm in diameter and 150 mm in length) and was suirable using solvent A (0.05% OF F) and solvent (isopropanol: acetonitrile = 7:3, 0,02% F) 30 min linear gradient from 1% to 50% when the speed of the current of 0.25 ml/min and the column temperature 30oC. in This way, were made by peptide mapping (see Fig.4). The resulting peptide fragments were extracted and subjected to cleavage by Erdman using amino acid sequencing machine with a gas phase (PPSQ-2, manufactured Shimadzu Corp. ). After that, each amino acid is obtained in turn from the sequencing machine, N-end of which was connected with PTH identified using C18 column chromatography with reversed phase using isocratic elution. The results are presented in table.II.

The sequences presented in table.II, the sequence shown in brackets in N-ends, are amino acid residues that can be deduced from the directed enzymatic cleavage.

It included the following databases:

.Entrez Release 6 database

.NCB1-GenBank, August 15, 1993 (Release 78.0)

.EMBL, July 15, 1993 (Release 35,0 plus updates)

.DDBL, July 15, 1993

.SWISS-PROT, April, 1993 (Release 25,0)

.PIR, June 30, 1993 (Release 37,0)

.PDB, April, 1993

.PRF, May, 1993

.dbEST, July 15, 1993 (Release 1,10)

.U.S. and European Patents

As a result, the sequence (K) DSFLADVK AP12 fully coincided with the internal sequence KDSFLADVK rat corticosteroidresponsive predecessor globulin (CBG) [PIR data base (accession No A40066; Smith and Hammond; "Rat corticosteroidresponsive globulin: primary structure and mRNA levels in the liver under different physiological conditions", Mol. Endocrinol., (1989), 3, 420-426].

Further detailed study revealed that the sequence KQYYESE (SEQ 1D No 193) with high similarity is lesti. In rat G sequence corresponding to the sequences A and A, are connected to each other, forming an internal amino acid sequence KDSFLADVKQYYESE (SEQ 1D No 190).

With regard to the amino acid sequences of other fragments than AR and AR, were not detected protein or gene similarity which could be discussed.

Example 3. Analysis of biological characteristics of the solid obtained from blood plasma thrombocytopenic rats

(1) In the test system rat CFU-MK (liquid culture system)

As a typical example, Fig.5 shows a graph of the dependence of the response on the dose in the case of the use of samples of solid, partially purified from plasma thrombocytopenic rats (TRO-active fraction F2 from the column YMC PackProtein-RP, described in the stage of purification (8) of example 1-2). When periodic observation of cultured cells under the microscope revealed the generation and maturation of megakaryocytes, namely the increase in cell size, possibly together with increased numbers of megakaryocytes. As a particularly significant changes in the formation of megakaryocytes was detected on day 4, which was the last day of cultivation (they were hard identifiable on-1052, 1987) or by the formation of prothrombotic (Torr et al., Blood, vol. 76, pp. 912-924, 1990), who consider the structure of the precursors of platelets, further differentiated from Mature megakaryocytes, and these prothrombotic consider the final stage of differentiation of megakaryocytes, the observed still in vitro. Since this morphological change was observed with high frequency when using only samples of the TORUS, it is possible that this factor alone can stimulate the proliferation and differentiation of CFU-MK, can produce Mature megakaryocytes and may eventually release platelets.

(2) In the test system of counting colonies

When the test sample TRO, partially purified from plasma thrombocytopenic rats, using the test system of counting colonies using unseparated bone marrow cells of rats, cells each stage separation/preconcentration or Gp11b/111a+CFU-MK fraction of the solid obtained from the plasma of rats, stimulated the formation of colonies of megakaryocytes. Compared with colonies of megakaryocytes induced by other cytokines, such as IL-3, murine GM-CSF or human EPO induced TRO colony of megakaryocytes was different in that each colony consisted of some m is s in since the TRO did not cause the formation of colonies or caused a small formation of colonies of other cell lines, Meg-CSF activity TRO can be considered as megakaryocyte-specific. On the basis of these facts it is obvious that TRO has different biological properties compared with the properties of other cytokines, such as rat IL-3, murine GM-CSF and human EPO, and manifests a unique Meg-CSF activity.

Sample TRO, partially purified from plasma thrombocytopenic rats also showed Meg-CSF activity on CD34+DR+cell fractions derived from cells of human bone marrow or blood cells of the spinal cord of man, and induced a significant number of colonies of human megakaryocytes, suggesting that this factor has no species specificity.

Example 4. Specialization rat TRO producing cells

(1) Screening of producing the solid organs of rats

Screening and specialization producing TRO organs of rats was performed in order to provide a source of mRNA for use in the cloning of the gene SRAW rats. First, from rats made thrombocytopenic by introducing the P55 antibodies, periodically took bone MH obtained culture supernatant was tested using the test system rat CFU-MK. However, this initial attempt did not give clear results. Then made further attempt cultivation of liver cells (hepatocytes) received collagenase perfusion of the disease thrombocytopenic rats, induced by the introduction of the P55 antibodies with tailored messages indicating the relationship between the liver and the formation of SRW in rats (Siemensma et al., J. Lab. Clin. Med., vol.86, pp.817-833, 1975).

When applying the obtained culture supernatant to the column WGA-Agarose and subsequent fractionation of the adsorbed fraction on a Vydac phenyl column with reversed-phase was found very similar activity at the same position obtained from the plasma of rats TRO in the test system rat CFU-MK. Weak, but the same activity was detected in the culture supernatant of normal rat hepatocytes. These results suggest that the liver is probably one of producing the solid bodies.

(2) Screening of producing TRO rat cell lines

Based on the above results were screening the rat producing TRO cell lines. First, each of 20 obtained from rat liver cell lines were cultured in the appropriate subculturally W the same corresponding medium with 5% FCS and the cultivation was continued for 3 days. When each of the obtained culture supernatant was partially purified according to the procedure described in stage (1) to study the availability of education TPO, TPO activity was clearly detected in three derived from hepatic parenchymal cells of rat cell lines, namely ACI-R8994 cells (ATSC CRL 1602, Becker et al., "0ncodevelopmental Gene Expression, ed. by Fishman and Sell, Academic Press, NY, pp.259-270, 1976; purchased from Dainippon Pharmaceutical Co. Co., Ltd.), H4-11-E cells (ATSC CRL 1548, Pitot et al. Nat. Cancer Int. Monogr., vol.13, pp. 229-245, 1964; purchased from Dainippon Pharmaceutical Co., Ltd.) and NTS cells (Thompson et al. , Proc. Natl. Acad. Sci, USA, vol. 56, pp. 296-303: 1966, purchased from Dainippon Pharmaceutical Co., Ltd.).

(3) a Detailed analysis of TPO activities in McA-RH8994 cells, H4-11-E cells and NTS cells

Biochemical and biological properties TRO-active proteins secreted from these three rat cell lines investigated in detail and compared with the properties obtained from the plasma of rats TRO.

McA-RH8994 cells suspended in alpha-MEM(-) liquid culture medium containing 10% FCS and transferred into a plastic flask microculture (175 cm2) at 1106cells per flask. After 3 days of culturing at 37oWith the incubator with CO2(5%) this medium was replaced Kul is moralnego supernatant. H4-11-E cells suspended in modified according to the method of Dulbecco environment Needle (liquid medium) (glucose 4.5 g/l, then this environment is referred to as DMEM) containing 10% FCS and transferred into plastic flasks (175 cm2) at 5105cells per flask. After 3 days of culturing at 37oC in an incubator with 5% CO2the medium was replaced with IMDM medium containing 5% FCS, and the cultivation was continued for another 3 days to get the resulting culture supernatant. Also NTS cells suspended in D liquid culture medium containing 5% FCS and transferred into plastic flasks (175 cm2) when 2,5105cells per flask. After 3 days of culturing at 37oC in an incubator with 5% CO2the medium was replaced with culture medium IMDM containing 5% FCS, and the cultivation was continued for 3 days to extract obtained culture supernatant.

Using a 2 l thus obtained culture supernatant from each of these three cell lines was performed partial purification derived from cell lines of solid radwaste in accordance with the procedure of cleaning SRW from XRP described in example 1-2. The results obtained are described below.

First, each culture supernatant was concentrated at the ri using column Sephadex G-25. The obtained eluate was applied to a column of Q-Sepharose FF column was washed in 20 mm Tris-model HC1 (pH 8.0) and then adsorbed fraction was suirable 20 mm Tris-model HC1 (pH 8.0) containing 175 mm NaCl. Elyuirovaniya thus the fraction was applied to a WGA-Agarose column, the column washed with PBS and adsorbed fraction was suirable 20 mm sodium phosphate (pH of 7.2) containing 0.2 M GlcNAc and 0.15 M NaCl. The obtained eluate was applied to a column of TSK-gel AF-BLUE 650MH, the column washed with 20 mm sodium phosphate (pH of 7.2) containing 1 M NaCl, and then the adsorbed fraction was suirable 2 M NaSCN. The obtained eluate was applied to a column of Phenyl-Sepharose 6 FF/LS, the column was washed with 50 mm sodium phosphate (pH of 7.2) containing 1.5 M ammonium sulfate, followed by washing 0.8 M ammonium sulfate, containing 36 mm sodium phosphate, and then the adsorbed fraction was suirable 20 mm sodium phosphate (pH of 7.2). Thus obtained fraction was concentrated and then fractionally using column Vydac Protein C4 reversed-phase (The Separation Group, cat. TR, diameter 1 cm, height 15 cm). I.e., this sample was applied to a C4 column, pre-equilibrated to 20%, and then elution was performed 90 minutes linear gradient from 20% to 40% at flow rate 1 ml/min, with 0.1% F as a developing solvent a and 1-propane is s, derived from these cell lines was blueraven at a concentration of 1-propanol 30 to 43%.

In the measurement of SRAW activities in samples of each stage of purification using the test system rat CFU-MK results showed that TPO activity of these 3 cell lines gave similar distribution activities obtained from XRP TRO (comparative example 1-2). The relative activity output activity, etc. at each stage of purification, measured in the test system rat CFU-MK, shown in table 2. In addition, in the measurement of TPO activity in the eluates from the final column reversed-phase test system rat CFU-MK TRO activities 3 cell lines and the activity obtained from XRP TRO were found in the same peaks. When conducting a test by counting colonies TRO-active fractions from the column with reversed phase with the use of unattached cells obtained at the stage of exhaustion attachment for separation and concentration of rat 11b/street 111A+CFU-MK, distribution elution, MEG-CSF activity were very similar to the distributions of TPO activity measured in the test system rat CFU-MK (see table 3).

In addition, like received from XRP TRO, each of TPO activities of these 3 cell Linah active fractions from the column with reversed phase were subjected to SDS-PAGE according to the procedure described in example 1-2. When extraction of protein from the resulting gel and measuring the activity of TPO in the test system rat CFU-MK was found that the average mol. the mass obtained from XRP TRO obtained from cells ACI-R8994 TRO and obtained from cell H4-11-E TRO were 17000-22000, 33000-39000 and 31000-38000, respectively, as obtained from the NTS cells TRO found average mol. mass 17000-22000 and 28000-35000.

Thus, these results showed that TPO proteins produced McA-RH8994 cells, H4-11-E cells and NTS cells, equivalent obtained from XRP TRO on their biological properties, although their biochemical properties are slightly different, particularly in relation to mol.mass. A remarkable discovery in this invention is the possibility that the molecule TRO-active protein contained in the blood, may be the product of a partial splitting in its specific or disordered website in producing its cells or after its secretion from the producing cells. It also suggests the possible existence of genes TRO (mRNA and cDNA) having a different length.

Example 5. Designing expressing vector (pEF18S) for cDNA library

Vector, which can be easily integrated DNA fragment and which is ionirovanii and expression of cDNA TRO. I.e. expressing vector pEF18S was constructed from expressing vector pME18S by substitution of SR promoter the promoter of the elongation factor I (EFI), which is known as the promoter, causing high expression (see Fig.6). The promoter of the elongation factor I was obtained by partial cleavage of 1 µg expressing vector pEF-BOS (Mizushima et al., Nucleic Acids Res., vol. 18, p. 5322, 1990) Hind111 restriction enzymes and EcoRI, after which the cleavage product was subjected to electrophoresis in 2% agarose gel (FMC BioProducts) for isolation of DNA fragment of about 1200 p. N. and purification of the DNA fragment with the use of the kit for purification of DNA Prep-A-Gene (Bio-Rad Laboratories, Inc.; set for use in the selective purification of DNA, which is accomplished by adsorption of DNA porous matrix of silica, based on the procedure reported by Willis et al., in Bio Tehniques, vol.9, pp.92-99, 1990), 100 ng of the thus obtained DNA fragment ligated with 50 ng expressing vector pME18S (Liu et al., Proc. Natl. Acad. Sci. USA, vol.90, pp. 8957-8961, 1993), which was previously split HindIII and EcoRI. Cells commercial strain-host, highly competent strain d b 5 E. coli (Toyobo Co., Ltd.; competent E. coli strain obtained by the modified method of Hanahan et al., J. Mol.Biol. vol.166, pp. 557-580, 1983), transformed with the and randomly and from each colony was purified plasmid DNA. In General received 10 clones containing target plasmid (pEF18S), analysis of distributions splitting restrictase these DNA molecules. After that, a clone selected from them, were cultured to obtain large quantities of the plasmid DNA.

Purification of plasmid DNA was performed basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Ie clone. EF18S obtained as described above were cultured overnight at 37oWith 50 ml of medium L (1% Bacto-Tipton, 0,5% t-yeast extract and 0.5% NaCl) containing 50 μg/ml ampicillin, and the cells were collected by centrifugation and suspended in 4 ml TED-lysozyme (25 mm Tris-HCl, pH 8, 10 mm EDTA, 50 mm glucose, 0.5 lysozyme). To it was added 8 ml of 0.2 N NaOH/1% SDS (solution) and then 6 ml of 3 M solution of potassium /5 M acetate for thorough suspension of cells. After centrifugation of this suspension obtained supernatant liquid was treated with a mixture of phenol/chloroform (1:1), mixed with the same volume of isopropanol, and then centrifuged. The precipitate was dissolved in TE solution (10 mm Tris-HCl, pH 7.5, 1 mm EDTA) and treated with RNase and then with a mixture of phenol/chloroform (1:1) followed by precipitation with ethanol. Received sieges the Noy concentration 0,63 M and 7.5%, respectively. After centrifugation the precipitate was dissolved in THE solution and precipitated with ethanol. In this way received approximately 300 μg of plasmid DNA. 100 μg of this DNA was completely digested with restrictase EcoRl and Notl and subjected to electrophoresis on 0.8% agarose gel (izgotovliaemye F BioProducts) and extracted thus fragments of the vector was purified using the kit for purification of DNA Prep-AGene (Bio-Rad Laboratories, Inc.), receiving approximately 55 μg of plasmid DNA, which was used in the subsequent construction of cDNA library.

Example 6. Purification of mRNA from ACI-R8994 cells

ACI-R8994 cells, found relatively high activity in the test by counting colonies of example 4, was selected as the material for use in cloning cDNA SRAW rats and subjected to the following experiments.

The allocation of total RNA was performed basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). I.e. McA-RH8994 were grown to confluence in 15 culture plates 90 mm in diameter. After removal of the liquid medium of the cells in each Cup carefully suspended in 0.8 ml of 5 M solution of guanidine (5 M guanidinium, 5 mm sodium citrate (pH 7.0), 0.1 M-mercaptoethanol, 0.5% carcaillet sodium) and the rum. The resulting mixture, which has become viscous due to the destruction of cells were subjected to approximately 20 repetitions suction/ discharge syringe 20 ml, equipped with a needle 21G, until then, until the mixture was no longer viscous. 18 ml of 5.7 M CSC1-0.1 M EDTA (pH 7.5) was used as a cushion in polyallomer centrifuge tube for use in the rotor SW28 Beckman, and the above mixture in an amount of about 20 ml was carefully layered on the pillow so that the layers have not been disturbed and the tube was almost full. Thus obtained tube was centrifuged for 20 hours at 25000 rpm at 20oC. the precipitate was washed twice with a small amount of 80% ethanol, dissolved in TE solution was extracted with a mixture of phenol/chloroform (1:1) and then precipitated with ethanol with 1/10 volume of 3 M sodium acetate and 25 volumes of ethanol. In this way received approximately 2.5 mg of total RNA from approximately 108cells.

Purification of poly (A)+RNA from total RNA was performed using ligotexTM-dT30 (Super) (manufactured by Japan Synthetic Rubber/Nippon Roche; dT molecules immobilized on latex particles by covalent binding and purification of poly (A)+PHK can be achieved similar to how this is ug of poly(A) +RNA was extracted from approximately 500 µg total RNA.

Example 7. Construction of cDNA library rat

Double-stranded cDNA having a website recognition EcoRl at its 5'end, and website recognition Notl at its 3'end was synthesized from 5 μg poly (A)+RNA obtained as described in example 6, with the use of the kit for cDNA synthesis Time SaverTM(manufactured Pharmacia; kit for cDNA synthesis, based on a modified method of Okayama and Berg, Mol. Cell. Biol., vol.2, pp. 161-170, 1982) and DIRECTIONAL CLONING TOOLBOX (manufactured Pharmacia; a set of

primer 5'- AACTGGAAGAATTCGCGGCCGCAGGAA(T)18-3' (SEQ 1D No 15), containing the sequence of recognition Notl, for use in the synthesis of cDNA, and the adapter 5'- AATTCGGCACGAG-3' (SEQ 1D no 16) 5'-CTCGTGTGCCG-3' (SEQ 1N No 17) for use in the adding sequence recognition EcoRl). Synthesized thus cDNA ligated with 1.2 µg expressing vector pEF18S (see example 5), which was previously split EcoRl and Notl, and transformed 8.4 ml of the above-mentioned highly competent E. coli DH5 (manufactured by Toyobo Co., Ltd.). The resulting 5,3105the transformants.

Example 8. Receiving (cloning) cDNA fragment SRAW rats using PCR

530000 clones cDNA library Mca-RH8994 constructed in example 7, cultiv is Ali from the received cells, collected by centrifugation, using QIAGEN-tip 100 (manufactured DIAGEN; anion-exchange column with silica for purification of DNA). Received approximately 200 μg of plasmid DNA.

Synthesized two antisense nucleotide primer AP18-1R and IR-2R, which correspond to the amino acid sequence of the peptide fragment OR described in example 2, and two semantic nucleotide primer FI-1 and EFI-2, which correspond to the first intron of the factor elongation person plasmid vector pEF-18S (see Fig.6) used to obtain a cDNA library. The synthesis of these primers was performed using 394DNA/RNA synthesizer (manufactured Applied Biosystems, synth-based-cyanoethylidene the way), and the purification was performed using column ORS for the purification of synthetic DNA (manufactured by Applied Biosystems; column with silica gel, reversed phase for use in the purification of synthetic DNA having triteleia group). Each of the thus synthesized and purified DNA was dissolved in TE solution to a final concentration of 50 μm and stored at -20oTo their use. Synthetic oligonucleotides used in the following procedure, was synthesized and purified in the same of obrazu which included deoxyinosine, as reported Takahashi et al. (Proc. Natl. Acad. Sci. USA, vol. 82, pp.1231-1935, 1985).

Primers EFI-1 and EFI-2, consisting of 21 and 20 nucleotides, respectively, were synthesized based on the nucleotide sequences of the provisions 1491-1512 and 1513-1532 genomic sequence reported Uetsuki et al. (J. Biol.Chem., vol. 264, pp. 5791-5798, 1989). (see tab.III)

With 3 µg ACI-R8994 cDNA library plasmid as template, AR-1R (500 pmoles) and EFI-1 (100 pmole) as primers and reagents GeneAmpTMPCR with DNA polymerase AmpliTaqTM(manufactured Takagi Shuzo Co., Ltd., set of thermostable Taql polymers, reaction buffer and dNTP for use in PCR) PCR was performed using GeneAmpTMPCR system (Perkin-Elmer; Thermoblock for PCR (100 μl volume, heated at 95o2 min, 35 cycles, each cycle consists of denaturation at 95oC for 1 min, hybridization (annealing) at 40oC for 1 min, and synthesis at 72oC for 1 min, and a final incubation at 72oC for 7 minutes). To improve the specificity of the amplified thus DNA fragments further PCR was performed (100 μl volume, heated at 95oC for 2 minutes, 35 cycles, each cycle consists of denaturation at 95oC for 1 minute, Gib with 72oC for 7 minutes) using 1 μl of the obtained solution of the PCR reaction as template and EFl-2 (100 pmole) and AR-IR (500 pmoles) as primers.

Thus obtained reaction solution was subjected to electrophoresis in 2% agarose gel (izgotovlena FC BioProducts) for isolation of DNA fragment of approximately 330, etc., ad as the primary PCR product which was then purified using the above kit for purification of DNA Prep-A-Gene (manufactured by Bio-Rad Laboratories, Inc.) with the use of T4 DNA ligase (manufactured by Life Technologies), thus purified DNA fragment was subcloned into the vector pCRTM11 (a vector for use in THE cloning of PCR products, produced Invitrogen). Since thermostable polymerase used in PCR, has terminal transfersno activity, amplificatory DNA fragment was directly subcloned into the vector pCRTM11, which has a 5'-dT sticky end, using the property of this enzyme to add one deoxyadenylate acid to the 3'-end amplified using PCR DNA. Plasmid DNA was purified from 28 clones selected randomly from the obtained clones using QIAGEN-tip100 (DIAGEN), and their nucleotide sequences were determined with swill
Terminater Cycle Seguening Kit (Applied Biosystems; set for use in the determination of the nucleotide sequence with the use of fluorescent dyes based on dideoxy-fashion Singer and others (Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467, 1977). When the DNA fragment encoding three amino acid residue (Ile/Thr/Ser)-Val-Pro amino acid sequence OR shown above, adjacent to the primer AP8-2R, was collected from the thus obtained DNA fragments and sequenced its full length, and it contained the cDNA of 261, etc., ad Position 173-175 this cDNA fragment encodes methionine and frame coding coincided with the amino acid frame AR. Since the sequence forewarned is in position 173-175 DNA fragment, coincided with the Kozak sequence (Kozak, M., Cell, vol. 44, pp. 238-292, 1986), one might think that this DNA fragment consists of the district of translation initiation encoding N-terminal protein SRAW rats. This DNA fragment was named A1. The nucleotide sequence of the A1 fragment comprising a vector sequence, and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D No 1), attached here.

Example 9. Screening of the cDNA clone SRAW rats using PCR

The cDNA library was divided into pools Pribylina, and then was extracted plasmid DNA using an automated device for the extraction of plasmid DNA P1-100 (VER-3,0, Kurabo Industries, Ltd. automated device for extraction of plasmid DNA, based on a modified method of alkaline SDS method described in Molecular Cloning, Sambrook et. al., Cold Spring Harbor Laboratory Press, 1989). Separately synthesized and purified following two oligonucleotide-based fragment A1 cDNA.

5'-CGAGGGTGTACCTGGGTCCTG-3' (SEQ 1D No 31) (semantic consistency provisions 1-17 sequence SEQ 1D No 1; CGAG denotes adapting the sequence 5'-CAGAGTTAGTCTTGCGGTGAG-3' (SEQ 1D No 32) (the antisense sequence of the provisions 212-232 sequence SEQ 1D No 1).

Using 1/30 volume of each sample of plasmid DNA obtained as described above as a matrix, synthesized so oligonucleotides as primers and reagents GeneAmpTMPCR with AmpliTaqTMDNA polymerase (manufactured by Takara Shuzo Co., Ltd.) PCR was performed using GeneAmpTMPCR System 9600 (PERKIN-ELMER) (a total of 30 cycles, each cycle consists of denaturation at 94oC for 30 seconds, hybridization with 66oC for 30 seconds, and synthesis at 72oC for 1 minute). The result found silanov for extraction of plasmid DNA and carrying out PCR in the manner described has detected a specific band in 3 of 100 subpool. Further separation of these subpool on pools of 40 clones and holding the same screening process specific band was detected in 3 out of 100 tested pools. One of the pools of candidates were cultured on L tablet (LB medium containing 15% agar) supplemented with 50 μg/ml ampicillin and each of the formed colonies were subjected to extraction of plasmid DNA and PCR as described. The positive band was detected in 2 out of 100 tested clones.

Example 10. Sequencing of cDNA SRAW rats

Purification of plasmid DNA was performed basically in accordance with the procedure described in Molecular Cloning (Sambrook et. al., Cold Spring Harbor Laboratory Press, 1989). Each of the two obtained clones of example 9 were cultured overnight in 50 ml LB medium containing 50 μg/ml ampicillin, and approximately 300 μg of plasmid DNA was obtained after purification as described in example 6.

Thus obtained plasmid DNA sequenced according to the method described in example 8 using a set of Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems) to determine the complete nucleotide sequence containing the A1 fragment. In the nucleotide sequence of these two clones was identical with each other, svideteli pEF18S-A2. Its nucleotide sequence and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D No 2).

The sequence represented in the list of sequences (SEQ 1D No 2) has distinct characteristics. The sequence in the direction 5'-3' from 172, etc., N. 5'-netransliruemye region encodes the amino acid sequence rich in hydrophobic amino acids, which is thought to be a signal sequence for secretion of the protein, consisting of 21 amino acids, beginning with methionine. This protein contains a 126 amino acid residues. 3'-netrakona sequence contains 1025 nucleotides and a poly a-tail after the termination codon (TAA). This protein contains a sequence that corresponds to amino acid sequence OR analyzed in example 2 (amino acid residues 1-12 in SEQ 1D No 2), but does not contain consensus sequences for N-glycosylation. The nucleotide sequence 1624-1629, located near the end of the 3'-noncoding sequence differs from the consensus sequence and is, apparently, a potential polyadenylation sequence.

Ve is In National Institute of Bioscience and Human Technology, Agency of Industrial Science and Techology, Ministry of International Trade and Industry, Japan.

Example 11. Expression of cDNA SRAW rats in COS 1 cells and confirmation of TPO activity

Transfection of plasmids pEF18S-A2 COS 1 cells was carried out as follows by a slight modification of DEAE-dextranomer the method comprising treatment with chloroquine (Sompayrac et al., Proc. Natl. Acad. Sci. USA, vol. 78, pp. 7575-7578, 1981; Luthman et al., Nucl. Acids Res., vol. 11, pp. 1295-1308, 1983). COS 1 cells (ATSC CRL 1650) suspended in the above-described environment DE containing 10% FCS was placed in a plastic Cup for tissue culture (100 mm) and cultured at 37oC in an incubator with 5% CO2up until the cells had not reached the stage of approximately 40% confluence. Separately, plasmid pEF18S-A2 (10 μg) dissolved in 30 μl of HBS (21 mm HEPES, 145 mm NaCl, pH 7,1) was added to 4 ml of culture medium DMEM, was added to 500 μg/ml DEAE-dextran (Pharmacia), 80 μm chloroquine (Sigma Chemical Co.), 8% (about. /about.) HBS and 9% (about./about.) Nu - serum (Collaborative Research, Inc.). Thus prepared mixture was added to cultured COS 1 cells, which were washed twice in culture medium, DMEM, and the cultivation was continued for 5 hours in an incubator with 5% CO2. Thereafter, the culture supernatant in the Cup was removed by suction and the remaining Cup of the Ali at 37oC for 3-5 days in an incubator with 5% CO2to extract obtained culture supernatant.

Thus obtained culture supernatant were dialyzed extensively against culture medium IMDM and evaluated in the test system rat CFU-MK. TPO activity was detected in a dose-dependent form in the culture supernatant of COS 1 cells, in which expressives plasmid pEF18S-A2 (Fig.7). As in the case with derived from RP TRO, many megakaryocytes formed elongated cytoplasmic education on the 4th day of cultivation. In contrast, TPO activity was not detected in the culture supernatant COS 1 cells, which were introduced only plasmid pEF18S (Fig. 7). In M-e test system activity proliferation of M-e cells was also found depending on the dosage form in the culture supernatant of COS 1 cells, in which expressives plasmid pEF18S-A2, but not in the culture supernatant of COS 1 cells, in which expressives plasmid pEF18S. These results show that A2 contains cDNA encoding a protein having TPO activity.

Then, a sample of partially purified TRO was prepared to test the ability of this TRO activity to reinforce established Bassington culture medium, which was added 0.2 mg BSA, receiving approximately 5,8 l serum-free culture supernatant. To it was added protease inhibitor pAPSF to a final concentration of 1 mm and the mixture was filtered using a 0.22 μm filter. To 5793 ml of the obtained filtrate (protein concentration 0,229 mg/ml, total protein 1326 mg, the relative activity 1000 total activity 1326000) was added to 0.85 mole of NaCl per 1000 ml (288 g), getting 5849 ml of a solution containing 0,822 M NaCl. Thus obtained solution was applied at a flow rate of 7 ml/min at a column TSK-gel AF-BLUE 650 MN (Tosoh Corp., cat. 08705, diameter 5 cm and height 6 cm), which was pre-equilibrated to 20 mm sodium phosphate (pH of 7.2) containing 1 M NaCl. After applying samples of approximately 7900 ml of the eluate was passed through the column by elution 20 mm sodium phosphate (pH of 7.2) containing 1 M NaCl. This eluate was collected together and concentrated using ultrafiltration devices (Omega Ultrasette, nominal clipping mol. mass 8000), manufactured Filtron), receiving passing through the column fraction F1 (460 ml, a protein concentration of 1.11 mg/ml, total protein 973 mg, the relative activity of 16.3. Then eluting the solution was replaced with 2 M NaSCN and elyuirovaniya thus absorbed TSK-gel AF-BLUE 650 MN TRO-active diameter, Amicon Corp.). Total protein in this TRO-active fraction F2 was 12.5 mg and the yield of protein F2 at this stage was 0.62 percent. Then F2 was applied on the column HiLoad 26/60 Superdex 200 pg (Pharmacia Biotech, cat. 17-1071-01, the diameter of 2.6 cm and height 60 cm), and showed a 20 mm sodium acetate (pH 5.5) containing 50 mm NaCl, at a flow rate of 1 ml/min After the beginning of the manifestation of TPO activity was detected in the eluates in the range from 194 to 260 ml after application. These eluate United, getting a Superdex 200 pg TRO active fraction F2 (66 ml, protein concentration 0,112 mg/ml, total protein 7,41 mg, the relative activity 142860 total activity 1058600). Then, the prepared buffer A (20 mm sodium acetate, pH 5.5) and buffer B (20 mm sodium phosphate, pH of 7.2, containing 500 mm NaCl) and TRO-active fraction was applied at a flow rate of 1 ml/min on a strong cation exchange column RESOURCE S (Pharmacia Biotech, cat. 17-1178-01, of 0.64 cm in diameter and a height of 3 cm), which was pre-equilibrated with 100% A. thereafter, elution was performed at a flow rate of 0.3 ml/min, 40 min linear gradient from 100% a to 100% Century When analyzing the activity of TPO activity found in a wide range of eluates (from 5% to 32%). These TRO-active eluate were combined and concentrated using ultrafiltration devices with MIND 3 membrane (diameter 25 mm, Amicon Corp. ), Paluch, total activity 558600).

A sample of partially purified TRO was administered subcutaneously for consecutive 5 days (474 μg (100 μl/mouse/day) ICR male mice (9 weeks of age), number of platelets which were measured on the day before the introduction. As a control the same way introduced BSA (200 μg (100 μl)/mouse/day) or buffer that is used as the solvent partially purified TPO (100 μl/mouse/day). The next day after the last injection had collected the blood from the heart of each mouse to measure the number of platelets using hemocytometer (F800, Toa Iyo Denshi). In mice that were injected partially purified TPO, watched the number of platelets, about 2.14 times more compared with the number of platelets before the introduction. When compared with control groups in the number of platelets in mice, which were injected partially purified TPO, were approximately of 1.74 times higher than the number of platelets in receiving BS mice and approximately 1.90 times higher than in the receiving buffer mice. These results showed that TPO enhances the formation of platelets in vivo. In addition, since the number of immunosuppressive acidic protein (1AP), known as the acute phase protein mice were not increased in mice that were administered castii protein acute phase.

Example 12. Detection of mRNA TRO in the tissues of rats

For localization expressroute mRNA TRO tissue in the body of the rat RNA was extracted from various tissues of the rat. A total of 6 rats were subjected to x-ray irradiation as described in example 1, and various tissues (brain, thymus, lungs, heart, spleen, intestine, kidney, testes, and bone marrow cells) were cut from these rats at the 11th - 14th day after irradiation and immediately frozen in liquid nitrogen. Extraction of total RNA was performed using the reagent for RNA extraction ISOGEN (manufactured Wako Pure Chemical Industries, Ltd.). The number of ISOGEN was determined according to the weight of the frozen tissue sample and the sample with the added reagent was treated with a homogenizer (PhyscotronRNS-60, N1T1-ON Medical & Physical Instruments MFG.Co., Ltd) to the complete destruction of tissue (approximately 45-60 seconds at 10,000 rpm). The tissue homogenate was then subjected to extraction of total RNA with the use of procedures based on the way with a mixture of sour guanidine phenol-chloroform (Chomczynski et al. (Anal. Biochem., vol.162, pp. 156-159, 1987). The result was obtained 1,1-5.6 mg total RNA from the respective tissues.

Using OligotexTM-dT30 (Super) (manufactured Japan Synthetic Rubber/Nippon Roche) 20 μg of poly(A)+RNA was purified approximately and the Li(A)+RNA obtained from each tissue. I.e., 1 μg poly (A)+RNA was dissolved in 10 μl of sterile water, incubated at 70oC for 15 minutes and then quickly cooled. To this solution was added 75 pmoles random primer (Takara Shuzo Co., Ltd.), 10 E of RNase inhibitor (Boehringer-Mannheim Corp.), 50 mm Tris-Hcl (pH 8.3), 75 mm KCl, 3 mm gl2and 200 E Super ScriptTM11 (reverse transcriptase made Life Technologies). The resulting solution (total volume 20 μl) were incubated at 37oC for 1 hour and the resulting reaction solution was incubated at 70oC for 10 minutes to inactivate the enzyme was stored at -20oTo use.

New primers for R synthesized based on the cDNA sequence SRAW rats, obtained in example 10. The sequence of the synthesized primers were as follows.

htra-1: 5'-CCT GTC CTG CTG CCT GCT GTG-3' (SEQ 1D No 33) (position 347-367 in SEQ 1D No 2).

rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC-3' (SEQ 1D No 34) (antisense primer corresponding to the provisions 1005-1025 in SEQ 1D No 2).

Using 1/10 volume of each solution of the synthesized cDNA as the template, 1 μm of each of the synthesized GTA-1 and rTPO-N as primers and GeneAmpTMPCR Reagent Kit with AmpliTaqTM<600 (PERKIN-ELER) and heated at 95oC for 2 minutes, repeat on the whole 30 cycles, each cycle consisted of denaturation at 95oC for 1 min, hybridization at 57oC for 1 min, and synthesis at 72oC for 1 minute followed the final incubation for 7 minutes. During electrophoresis the resulting reaction solutions on 2% agarose gel (FC BioProducts) and the test of the amplified bands bands that seemed to be specific for mRNA TRO were found in gels derived from brain, liver, small intestine and kidney. These results indicate that the expression of mRNA TRO in rats occurs in the tissues of these organs, although its quantitative expression cannot be evaluated. While discussing the possibility of the existence of such a way of expression in humans, and the results of example 4, it can be assumed that the liver is a suitable starting material for obtaining cDNA TRO person.

Example 13. Construction of cDNA library derived from normal human liver

Based on the results of example 12, the liver was selected as the starting material for use in cloning cDNA TRO person. In the same manner as described in example 7, the double-stranded cDNA, Keysaney from normal human liver poly(A)+RNA (manufactured by Clontech; the product was extracted on the basis of the method with a mixture of sour guanidine-phenol-chloroform Chomczynski et al.), using Time SaverTMcDNA synthesis Kit (kit) (manufactured Pharmacia) and DIRECTIONAL CLONING TOOLBOX (manufactured Pharmacia). Thus obtained cDNA ligated with 1.2 µg expressing vector pEF18S, which was split in advance EcoRl and Notl, and transformed 8.4 ml of Competent E. coli strain DH5 (Toyobo Co., Ltd.). The result was 1,2106the transformants.

Example 14. Receiving (clone) the cDNA fragment TRO person using PCR

Using a random primer first chain cDNA was synthesized from 1 µg commercial obtained from normal human liver poly(A)+RNA (manufactured by Clontech). I.e., 1 μg poly(A)+RNA was dissolved in 10 μl of sterile water, incubated at 70oC for 15 minutes and then quickly cooled. To this solution was added 75 pmoles random primers (Takara Shuzo Co., Ltd.), 10 E of RNase inhibitor (Boehringer-Mannheim Corp.), 50 mm Tris-Hcl (pH 8.3), 75 mm KCl, 3 mm MgCl2200 E. reverse transcriptase Super ScriptTM(manufactured by Life Technologies). Thus obtained solution (total volume 20 μl) were incubated at 37oC for 1 hour and the ri -20oTo use.

The primers for R synthesized based on the cDNA sequence of cDNA SRAW rats (SEQ 1D 2). The sequence of the synthesized primers were as follows.

rTPO-AIN: 5'-ATG GAG CTG ACT GAT TTG CTC -3'- (SEQ 1D 35) (position 173-193 in SEQ 1D 2).

rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC -3' (SEQ 1D 36) antisense primer corresponding to the provisions 1005-1025 in SEQ 1D 2).

Using 1/10 volume of a solution of the synthesized cDNA as the template, 1 μm of each of the synthesized thus rTPO-A1N and rTPO-N as primers and reagents GeneAmpTMPCR with DNA polymerase AmpliTaqTM(manufactured by Takara Shuzo Co., Ltd.) PCR was performed with a volume of 100 µl, using GeneAmpTMPCR System 9600 (PERKIN-ELMER) and heated at 95oC for 2 minutes, repeating 35 cycles consisting of denaturation at 95oC for 1 min, hybridization at 40oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes.

Thus obtained reaction solution was subjected to electrophoresis in 2% agarose gel (izgotovlena FMC BioProducts) for isolation of DNA fragment of approximately 620, etc., ad as the primary PCR product is tidow sequence of the thus purified DNA fragment was immediately determined by DNA sequencing machine A (Applied Biosystems) using the above set of Taq Dye DeoxyTMTerminater Cycle Sequening Kit (manufactured Applied Biosystems). The thus determined nucleotide sequence, except primerno, parts, and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D 3).

This DNA fragment, except primernih sequences has a length of 580, etc., N. When comparing this cDNA man found the homology of 86% with the nucleotide sequence of the cDNA of the rat, suggesting that this DNA fragment encodes part of the TRO person.

Example 16. Screening of the cDNA clone TRO person using PCR

The relevant TRO person primers for R synthesized on the basis of SEQ 1D 3. Sequence synthesized thus primers were as follows.

hTPO-I: 5'-TTG TGA CCT CCG AGT CCT CAG - 3' (SEQ 1D 37) (position 60-80 in SEQ 1D 3).

hTPO-j: 5'-TGA CGC AGA GGG TGG ACC CTC-3' (SEQ 1D 38) (antisense primer corresponding to the provisions 479-499 in SEQ 1D 3).

A library of human cDNA, constructed in example 13, amplified and divided into pools (each pool contained approximately 100,000 clones), were cultured overnight in 1 ml LB medium containing 50 μg/ml of ampicillin, and then they were extracted plasmid Dextrogyrate DNA was dissolved in TE solution.

Using 5% of extracted sample DNA as template, 1 μm of each of the synthesized oligonucleotides (hTPO-I and hTPO-j) as primers and a set of reagents for PCR GeneAmpTMwith AmpliTaqTMDNA polymerase (Tukara Shuzo Co., Ltd) PCR was performed in 20 μl volume using system R 9600 GeneAmpTM(manufactured by PERKIN-ELMER) (a total of 35 cycles, each cycle consists of denaturation at 95oC for 1 min, hybridization at 59oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes). The result of a specific band was detected in 3 of the applied 90 pools. One of the 3 pools were divided subpoly, each of which contained approximately 5000 clones, and a plasmid DNA was purified from 90 subclones for carrying out PCR using the described method. The result of a specific band was detected in 5 Subpolar. One of the 5 pools were divided subpoly, each of which contained 250 clones, and plasmodial DNA was extracted from 90 subpool, and specific band was detected in 3 Subpolar. One of the 3 pools were divided subpoly, each of which contained 30 clones, and a plasmid DNA was purified from 90 subpool for holding R described manner. In re the m LB tablet containing 50 μg/ml ampicillin, and each of the thus obtained 90 colonies were subjected to extraction of plasmid DNA and PCR as described. The result at the end was obtained clone HL34.

Example 16. Sequencing of cDNA SRAW man

Purification of plasmid DNA was performed basically in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Clone HL 34 were cultured overnight in 50 ml of L medium containing 50 μg/ml ampicillin, and the cells were collected by centrifugation and suspended in 4 ml of the above-mentioned TED-lysozyme solution. Thereto was added 8 ml of 0.2 N NaOH/ 1% SDS and then 6 ml of 3 M potassium/5 M acetate for a thorough suspension of cells. After centrifugation of this suspension obtained supernatant was treated with a mixture of phenol/chloroform (1:1), mixed with the same volume of isopropanol, and then centrifuged. The precipitate was dissolved in THE solution and were treated with RNase and then with a mixture of phenol/chloroform (1: 1) followed by precipitation with ethanol. The precipitate was again dissolved in THE solution, to which was added NaCl and polyethylene glycol 3000 to final concentrations 0,63 M and 7.5%, respectively. After centrifugation the precipitate was dissolved in THE solution and sadaputa plasmid DNA used in DNA sequencing machine A (Applied Biosystems) to determine its complete nucleotide sequence using q Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems). The thus determined nucleotide sequence and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D 4). In this case, as primers when determining the nucleotide sequence of the used oligonucleotides synthesized based on the nucleotide sequence of SEQ 1D 3, and synthetic oligonucleotides were designed based on the internal sequence obtained by the analysis of sekvencovany.

In the result, it was proved that the plasmid clone pEF18-S-HL-34 contains a cDNA fragment of 861 p. N. and contains vysokomolochnye sequence with amino acid sequences OR (number of amino acids 1-12 in SEQ 1D 4) and TR2/TR3 (amino acids 157-162 in SEQ 1D 4), analyzed in example 2. Apparently, this DNA fragment encodes an open reading frame beginning at position 25, but has no termination codon and contains a poly A-tail is similar to the sequence of 76 bases at its 3'end. Its amino acid sequence consisting of 253 amino acid residues, found 84% homology with the corresponding part of the cDNA SRAW rats (part of the amino acid follower is entom DNA the encoding part of the human cDNA that corresponds to the cDNA SRAW rats. Due to the absence of the termination codon and the presence of a poly a tail similar sequence at its 3'-end of the clone, apparently, is not complete cDNA, and is an artificial product obtained during the procedure of constructing the cDNA library.

Example 17. Expression of cDNA TRO person in COS 1 cells and confirmation of TPO activity

Transfection obtained as described plasmid clone pEF18S-HL-34 in COS 1 cells were performed according to the method of example 11. I.e., transfection was performed with 10 μg of plasmid DNA in DEAE-dextranomer the method comprising treatment with chloroquine. These COS 1 cells were cultured for 3-5 days and then received the supernatant.

The obtained culture supernatant were dialyzed extensively against culture medium IMDM and evaluated its activity in the test system rat CFU-MK. TPO activity was detected in a dose-dependent form in the culture supernatant of COS 1 cells, in which expressives plasmid pEF18S-HL-34 (Fig.8). After 4 days of cultivation many megakaryocytes formed elongated cytoplasmic protrusions. In contrast ü only plasmid pEF18S (Fig.8). In the test system with M-07e cells activity enhance proliferation of M-e cells was detected only in the supernatant of COS 1 cells, in which expressives plasmid pEF18S-HL-34 as a result of its transfection. These results showed that EF18S-HL34 contains a gene that encodes a protein having TPO activity. In addition, it was shown that TRO person acts on precursor cells of rat megakaryocytes (i.e., no species specificity).

Example 18. Expression of cDNA deletion type SRW person and confirm the TPO activity

Clone HL34 obtained in example 15 contained cDNA with a poly a tail like a continuous sequence at its 3'-end, which is not present in the cDNA SRAW rats, and so is, apparently, an artificial product of this experiment. As a consequence, received a cDNA molecule, which was demeterova sequence that is similar to the poly A-tail to check if the protein expressed by this cDNA deletion, TPO activity. Deletion cDNA was obtained using R. Sequences of primers used for R were such in which the site recognition by restriction enzyme was added to the 5'-end (coRI k hO5 and Notl and two stop codon TAA and TGA k h3).

Using 1 μg of plasmid DNA plasmid clone pEF18S-HL34 obtained in example 16, as a matrix, 10 μm of each of the synthesized hTO5 and h3 as primers and a set of reagents for PCR GeneAmpTMwith AmpliTaqTMDNA polymerase (Takara Shuzo Co., Ltd.,) PCR was performed with a volume of 100 μl using R system 9600 GeneAmpTM(PERKIN-ELMER) and was repeated a total of 15 cycles, each of which consisted of denaturation at 95oC for 1 min, hybridization at 65oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation for 7 minutes. Strip approximately 800 p. N. thus obtained was digested with restrictase EcoRl and Notl, purified, and then subcloned into the expressing vector pEF18S, which was pre-processed by the same restrictases. From the obtained transformants 5 clones containing DNA fragment of about 800 p. N., was selected to receive large quantities of plasmid DNA according to the procedure described in example 5. The full length of the amplified region of approximately 800 L. N. each plasmid were subjected to analysis of nucleotide sequence, receiving its full the

The resulting clone was named pHT1-231. Vector pHT1-231 carried by E. coli strain DH5 was deposited by the authors of the present invention 14 February 1994 under Deposit FERM BP-4564 In National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan. Transfection of the resulting plasmids in COS1 cells were performed according to the method of example 11. I.e., transfection was performed with 10 μg of plasmid DNA in DEAE-dextranomer the method comprising treatment with chloroquine. COS 1 cells were cultured for 3-5 days at 37oWith and then collected the supernatant.

Thus obtained culture supernatant were dialyzed extensively against culture medium IMDM and evaluated in the test system rat CFU-MK. TPO activity was detected in the culture supernatant of COS 1 cells, in which expressives plasmid pHT1-231 (Fig.9). On the 4th day of cultivation many megakaryocytes formed elongated cytoplasmic protrusions. In contrast, TPO activity was not detected in the culture supernatant of COS 1 cells, in which expressives only pEF18S (Fig. 9). M-07e test system activity enhance proliferation of M-e cells was detected only in the culture spank, which encodes a protein having TPO activity.

Example 19. Getting the 3'-terminal region of the cDNA TRO person using CR

Clone HL34 obtained in example 15, contained a cDNA having such poly A-tail sequence, on the basis of what was assumed to be at its 3'-terminal region was incomplete. Therefore, an attempt was made to obtain a full-sized 3'-terminal region using R. The following 4 types of primers 5 parties for PCR were synthesized based on the sequences identified in example 16:

hTPO-H: 5'-AGC AGA ACC TST HUNDRED GTC CTC - 3' (SEQ 1D 39) (position 574-594 in SEQ 1D 4).

hTPO-K: 5'-ACA CTG AAC GAG CTC CCA AAC-3' (SEQ 1D No 4) (provisions 595-615 in SEQ 1D 4).

hTPO-N: 5'-AAC TAC TGG CTC TGG GCT TCT-3' (SEQ 1D No 41) (position 660-680 in SEQ 1D 4).

hTPO-O: 5'-AGG GAT TCA GAG CCA AGA TTC-3' (SEQ 1D No 42) (position 692-712 in SEQ 1D 4).

The following primers 3'-side containing mixed nucleotides at 4 bases 3'-end were synthesized to amplify cDNA from the beginning And poly-part. Was synthesized anchor primer, without mixed nucleotide.

hTPO3mix: 5'-TAG CGG CCG C(T)17G GGG-3' (SEQ 1D No 43).

A AAA-3' (SEQ 1D No 44).

With TTT-3' (SEQ 1D No 45).

CCC-3' (SEQ 1D No 46).

hTPO3anchor: 5'-TAG CGG CCG C(T)11-3' (SEQ 1D No 47).

Second R was performed using 1/10 volume of a solution obtained in the first R, as a matrix, 20 μm hTPO-K primer and 10 μm hTPO3mix primer with a volume of 50 μl, heated at 96oC for 2 minutes and repeat total of 10 cycles, each of which consisted of thermal denaturation at 96oC for 1 min, hybridization at 63oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation in the second PCR, as a matrix, 20 μm hTPO-N primer and 10 μm hTP3mix primer with a volume of 50 μl, heated at 96oC for 2 minutes and repeat total of 10 cycles, each of which consisted of thermal denaturation at 96oC for 1 min, hybridization at 63oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes.

Fourth R was performed using 1/10 volume of a solution obtained in the third R, as a matrix, 20 μm hTPO-O primer and 10 μm hTPO3mix primer with a volume of 50 μl and heated at 96oC for 2 minutes, repeating total of 10 cycles, each of which consisted of thermal denaturation at 96oC for 1 min, hybridization at 63oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes.

Fifth PCR was performed using 1/10 volume of a solution obtained in the fourth PCR, as a matrix, 20 μm hTPO-O primer and 20 μm h3anchor primer with a volume of 50 μl by heating at 96oC for 2 minutes, repeat total of 10 cycles, each of which consisted of thermal denaturation at 96oIn techdude final incubation at 72oC for 7 minutes.

Thus obtained reaction solution was subjected to electrophoresis in 2% agarose gel (FMC BioProducts) for isolation of DNA fragment of approximately 600 p. N. as the primary PCR product which was then purified using the above kit for purification of DNA Prep-A-Gene (manufactured by Bio-Rad Laboratories, Inc.). The nucleotide sequence of the thus purified DNA fragment was immediately determined using a DNA sequencing machine 373 (Applied Biosystems) using the Taq Dye DeoxyTMTerminator Cycle Sequening Kit (Applied Biosystems). A specific nucleotide sequence and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D 5).

This DNA fragment has the nucleotide sequence encoding a 130 amino acids, starting with primer hTPO, followed by a sequence of 180 nucleotides at the 3'end (nucleotides after provisions 577 could not be determined). Amino acid sequence beginning with glycine, coincides with the amino acid sequence of the provisions 203-232 in SEQ 1D 4. The nucleotide sequence of positions 1-94 also coincides with the nucleotide sequence of positions 692-785 in SEQ 1D 4.

Note the config in example 15, contained such a poly A-tail sequence directly to its 3'-end of its open reading frame without termination codons and therefore was apparently unnatural product synthesis cDNA, a cDNA library was reconstructed using 5 µg commercial obtained from normal human liver poly (A)+RNA (drug, manufactured Clontech). Synthesis of cDNA was performed using SuperScriptTMLambda System for cDNA synthesis and Cloning Kit SuperScriptTM11 RNase H-(both manufactured by LIFE TECHNOLOGIES). Poly (A)+RNA was denaturiruet by heating and then added to 20 μl of reaction solution containing comprising the sequence of recognition Notl oligo dT as the primer included in the kit (50 mm Tris-Hcl, pH 8.3, 75 mm KCl, 3 mm Mgl2, 1 mm DTT, 1 mm dNTP mix, 200 E. SuperScriptTM11 RNase H-), followed by incubation at 37oC for 60 minutes. After synthesis of the second chain cDNA (2 hours incubation at 16oWith 150 μl of reaction solution containing 25 mm Tris-Hcl, pH 8.3, 100 mm KCl, 5 mm Mgl2, 250 μm dNTP mix, 5 mm DTT, 40 E. DNA polymerase 1E.coli, 2nd RNase H from E. coli and 10 E DNA ligase E. coli) was added 10 E T4 DNA polymerase, and the resulting mixture was incubated at 16oWith in the course the art of phenol/chloroform. The cDNA molecules that have a length of less than 400 p. N., removed using SizeSepTM400 rotating column (column to remove low molecular weight DNA attached to the kit for cDNA synthesis TimeSaverTM, Pharmacia). After adding EcoRl Adaptor (attached to the Directional Cloning Toolbox, Pharmacia) thus treated samples were digested Notl and again put on the same column to remove low-molecular-weight DNA. Synthesized thus double-stranded cDNA (1,3 g) having the sequence recognition EcoRl at its 5'end and sequence recognition Notl at its 3'-end ligated with expressing vector pEF18S, split pre-EcoRl and Notl, and then transformed 9.2 ml of Competent E. coli strain DH5 (Toyobo Co., Ltd.).

Thus obtained cDNA library of a human liver (hTPO-F1) contained 1,0106the transformants.

Example 21. Screening of the cDNA clone TRO from the library hTPO-F1 cDNA human liver

Primers corresponding cDNA TRO person, for R synthesized on the basis of SEQ 1D 3 and 6, are presented in the list of sequences. The sequence of the synthesized primers were as follows.

hTPO-1: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ 1D No 48) (position 60-80 in SEQ 1D 3).

hTPO-KU: 5'-AGG ATG GGT TG

Library hTPO-F1 cDNA human liver (1,0106transformed) constructed in example 20 was divided into 3 pool (pools 1 to 3), and these pools were frozen. PCR was performed using 2 μg of plasmid DNA obtained from each pool, as a matrix, and 1 μm of each of the synthesized oligonucleotides (hTPO-1 and hTPO-KU) as primers. Using a set of reagents for R GeneAmpTMwith AmpliTaqTMDNA polymerase (Takara Shuzo Co. Ltd. ) PCR was performed in a 100 µl volume using the PCR system 9600 GeneAmpTM(PERKIN-ELMER) (a total of 35 cycles, each of which consisted of denaturation at 95oC for 1 min, hybridization at 59oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes). As a result, the DNA fragment with the expected size amplified in the current case, when applied plasmid DNA obtained from the pool 3. Pool 3 then divide by subpoly, each of which contained 15,000 transformants, and these subpoly were cultured overnight in 1 ml of L medium containing 50 μg/ml of ampicillin, followed by extraction of plasmid DNA using an automated device for selection of plasmids P1-100. Extracted thus Decenium the same primers and conditions described above. As a result of DNA amplification with the expected size was detected in 6 of 90 pools. One of these positive pools were divided into subpoly, each of which contained 1000 clones were extracted plasmid DNA and spent R, as described above. The same was observed by DNA amplification. The density of the bands of DNA fragments by gel electrophoresis after a series of PCR became smaller, with further divisions into the pools, that, apparently, due to the low extraction of plasmid DNA due to the weak growth of the target clone. So another screening was performed by hybridization of colonies using the initial pool 3.

Pool 3 was divided into 100 LB agar plates 15 cm in diameter with the same amount of inoculum that each Cup has grown 4100 colonies. After receiving Cup replica from each of the inoculated thus cups one Cup-duplicates were cultured at 37oWith 6 hours to extract grown on the Cup colonies and extraction of samples of plasmid DNA. When performing PCR on these samples DNA, as described above, the amplification bandwidth, equivalent to the expected size was observed in one of the 100 subpool. From a Cup of this subpool were prepared by two filter-replica using BIODYNE/1.5 M NaCl for 10 minutes and 0.5 M Tris-HCl (pH 8.0)/1.5 M NaCl for 10 minutes in sequence, with the subsequent 30 min drying in air and then heated for 1 hour at 80oWith in a vacuum drying Cabinet. The resulting filters were washed in 6 x SSC (obtained by dilution of 20 x SSC source solution consisting of 175,3 g NaCl and 88,2 g Na-citrate dissolved in 1 l of water, pH 7.0) supplemented with 1% SDS. Prehybridization washed thus filters was performed by incubating them at the 42oC for 30 minutes with rocking in 30 ml of a reaction solution comprising 50% formamide, 5 x SSC, 5 x denhardt's solution (obtained from 50 x denhardt's solution containing 5 g of Ficoll, 5 g of polyvinylpyrrolidone and 5 g of fraction V bovine serum albumin in 500 ml of water), 1% SDS and 20 μg/ml DNA salmon sperm.

After prehybridization the reaction solution was exchanged with 30 ml of hybridization solution of the same composition, were mixed with probe, labeled [-32P] - CTP (Amersham) and then incubated with rocking at the 42oC for 20 hours. Labeled probe used in this experiment was an EcoRl/BamH1 fragment of plasmid pEF18S-HL34, which was obtained by the purification part of SEQ 1D 4 of its 5'-end to position 458 and tagging this purified part way arbitrary primers with Megaprime DNA Labelling System (kit, made Amersham, the DS solution at 42oC for 30 minutes and then in 0.2 x SSC/0,1% SDS solution at 42oC for 30 minutes. Then the filters were subjected to 16 hours of autoradiography at -70oWith using intensifying screen X-OMATTMthe AR5 film (Eastman Kodak). The result was observed by one signal, considered as positive. Colony, approximately corresponding to this signal, together with the original Cup and sown again on L agar Cup 10 see 50 colonies grown on this Cup, were cultured separately and their DNA samples were subjected to R using primers h-1 and h-KU under the above conditions. In the amplification band, equivalent to the expected size was detected in only one clone, which was named pHTF1.

Example 22. Determination of the nucleotide sequence of clone F1 cDNA SRAW man

Purification of plasmid DNA was performed mainly according to the procedure described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989). Clone pHTF1 were cultured overnight in 50 ml LB medium containing 50 μg/ml ampicillin. The resulting cells were collected by centrifugation and suspended in 4 ml of solution TED-lysozyme. Thereto was added 8 ml of 0.2 N NaOH/1% SDS and then 6 ml of 3 M potassium and 5 M ACE is treated with a mixture of phenol/chloroform (1:1), mixed with the same volume of isopropanol, and then centrifuged. The precipitate was dissolved in THE solution and were treated with RNase and then with a mixture of phenol/chloroform (1:1) followed by precipitation with ethanol. The precipitate was again dissolved in THE solution, to which was then added NaCl and polyethylene glycol 3000 to their final concentrations 0,63 M and 7.5%, respectively. After centrifugation the precipitate was dissolved in THE solution and precipitated with ethanol. In this way received approximately 300 μg of plasmid DNA pHTF1.

The sensitive plasmid DNA was used in the above-mentioned DNA sequencing machine A (Applied Biosystems) to determine its complete nucleotide sequence using Taq Dye DeoxyTMTerminator Cycle Sequening Kit (Applied Biosystems). The thus determined nucleotide sequence and deduced from it the amino acid sequence shown in the list of sequences (SEQ 1D 7). Oligonucleotides synthesized based on the nucleotide sequence of SEQ 1D 6 and the internal consistency obtained by the analysis of their sequencing, was used in the determination of the nucleotide sequence as primers.

In the result, it was confirmed that the clone F1 of seminarist 1-12 in SEQ 1D 7) and TR2/TR3 (number of amino acids 157-162 in SEQ 1D 7), analyzed in example 2. This DNA fragment, apparently, has a 5'- noncoding region of the 101 Foundation, an open reading frame consisting of 353 amino acid residues beginning with the methionine residue encoded at positions of nucleotides 102-104, and ending with glycine residue encoded at positions of nucleotides 1158-1160, next next termination codon (TAA) and 3'- noncoding region of 531 bases and sequence And poly-a tail of 30 bases. The amino acid sequence of the protein, which can be encoded by this open reading frame, fully coincided with the predicted amino acid sequence TRO person shown in SEQ 1D 6. The cDNA sequence pHTF1 was larger than the likely cDNA sequence shown in SEQ 1D 6, and contains 77 additional bases at the 5'end and 347 additional bases at the 3'-end sequence before the poly a-tail. Also, this nucleotide sequence differed from SEQ 1D 6 at three positions. I.e., A (position 84) And (position 740) and G (position 1198) in SEQ 1D 7 were S, T in SEQ 1D 6, respectively. Only the mutation at position 740 included in encoding the protein of the area, but this mutation did not cause the betrayal of the clusters at the bases currently unclear, analysis of the plasmid clone pHTF1 confirmed that protein TRO person contained 353 amino acid residue, including the signal sequence, consisting of 21 amino acids. Mol. the mass of the Mature protein without the signal sequence was identified as 35466.

Vector F1 in E. coli strain DH5 was deposited by the authors of the present invention 24 March 1994 under FERM BP-4617 at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Techology, Ministry of International Trade and Industry, Japan.

Example 23. Expression of clone pHTF1 cDNA TRO person in COS 1 cells and confirmation of TPO activity

Transfection of OS1 cells thus obtained plasmid clone pHTF1 were performed according to the method of example 11. I.e., transfection was performed with 10 μg of plasmid DNA DEAE-dextranomer the method comprising treatment with chloroquine. Transfetsirovannyh COS 1 cells were cultured for 3 days at 37oWith and collected culture supernatant.

The culture supernatant were dialyzed extensively against culture medium IMDM and evaluated in the test system rat CFU-MK. TPO activity was detected in a dose-dependent form in the supernatant of COS 1 cells, in which expressives plasmid pHTFl (Fig.10A). In contrast, T is a plasmid pEF18S (Fig.10A). Similar results were obtained in M-e test system. Supernatant of COS 1 cells, in which expressives plasmid pHTF1, significantly increased the proliferation of M-e cells dependent on the dose follows (Fig. 10b). These results showed that pHTF1 contains a gene that encodes a protein having TPO activity.

Example 24. Cloning of chromosomal DNA SRAW man

Cloning of chromosomal DNA TRO were performed using cDNA TRO person as a probe. The genomic library used in the cloning, was a gift from Professor T. Yamamoto from Gene Research Center, Tohoku University (library, reported Sakal et al. in J. Biol. Chem, 269, 2173-2182, 1994, designed by partial cleavage of the chromosomal DNA of a person by the restriction enzyme Sau3A1 and legirovaniem product partial cleavage with BamH1 site of phage vector Lambda EMBL3, manufactured Stratagene). Screening of this library using cDNA TRO person as a probe was performed basically according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Using E. coli LE392 as the owner of this library was inoculable on the Cup (15 cm) containing NZYM (10 g NZ amine, 5 g NaCl, 5 g Bacto - yeast extract, 2 g gSO47H2O and 15 g of agar in 1 l of water (pH 7.0) in takanah 18 cups using BIODYMETMA TRANSFER MEMBRANE (manufactured by PALL.). Denaturation of filters is performed by soaking them in 0.5 N NaOH/1.5 M NaCl for 10 minutes and then 0.5 M Tris-HCl (pH 8.0) /1.5 M NaCl for 10 minutes, followed by drying in air for 30 minutes and hot drying at 80oC for 1.5 hours in a vacuum thermostate. Prehybridization was performed by incubating the thus treated filter 42oC for 1 hour in 500 ml of a reaction solution comprising 50% formamide, 5x SSC, 5 x denhardt's solution, 1% SDS and 20 μg/ml DNA salmon sperm. For use as a probe a fragment of cDNA TRO person (number of provisions of the grounds 178-1025 in SEQ 1D 7) PCR amplified and purified, and the purified fragment was labelled 32R with a set for tagging random primers DNA (set for labeling DNA, produced by Takara Shuzo on the basis of the method of arbitrary primers described in Anal.Biochem. 132, 6-13, 1983). Sequences of primers used in this PCR were the following.

hTPO-1: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ 1D 50) (position 178-198 in SEQ 1D 7).

hTPO-N: 5'-AGG GAA GAG CGT ATA CTG TCC-3' (SEQ 1D 51) (antisense primer corresponding to the sequence positions 1005-1025 in SEQ 1D 7).

With the use of isotope labeled probe hybridization prehybridization solution. The resulting filters were washed 3 times in the solution of 2 x SSC / 0,1% SDS at room temperature for 5 minutes and then once in a solution of 0.1 x security standards /0,1% SDS at 68oC for 1 hour. Then the filters were subjected to autoradiography at -79oWith using intensifying screen and X-OMATTMAR5 film (Eastman Kodak). The result has been 13 positive signals. Plaques, approximately corresponding to each of the positive signals were collected from the original cups and inoculable again on NZYM cups (15 cm) in that the amount of inoculum that each Cup has formed 1000 plaques. Two filter replicas were prepared from each of the cups for conducting hybridization under the conditions described above. The positive signals were detected in all filters of the 13 groups. One plaque was removed from each of the cups to get ragovoy DNA according to the method of lysate cups, described in Molecular Cloning. Sample ragovoy DNA thus obtained of the 13 clones were checked for the presence of cDNA coding region using R using the following primers.

hTPO-L: 5'-GGC CAG CCA GAC ACC CCG GCC-3' (SEQ 1D 52) (position 1-21 in SEQ 1D 6).

hTPO-F: 5'-ATG GGA GTC ACG AAG CAG TTT-3' (SEQ 1D 53) (antisense primer corresponding to the placenta is stuudy sequence of the provisions of 1070 1090 in SEQ 1D 6).

When conducting PCR using these combinations of primers, 5 out of 13 clones, apparently, contained the full coding amino acids district, suggested from this cDNA. Chromosomal DNA molecules containing these 5 clones had a similar length of approximately 20 p. N., and found nearly the same distribution of fragments in the preliminary restriction analysis using restricted. Therefore, one of these clones (clone HGTl) was selected and analyzed by blotting on Southern. I.e., 1 ág DNA clone HGTl were digested completely with restriction enzyme EcoRl or Hind111 and inflicted by 0.8% agarose gel electrophoresis and bands on the gel were transferred to BIODYNETMA TRANSFR MEMBRANE (PALL). The resulting filter was air-dried for 30 minutes and annealed 2 hours at 80oWith vacuum Leche. Prehybridization was performed by incubating the thus treated filter 42oC for 1 hour in 50 ml of reaction solution comprising 50% formamide, 5 x SSC, 5 x denhardt's solution, 1% SDS and 20 μg/ml DNA salmon sperm. For use as a probe a fragment of cDNA TRO person (number of provisions of the grounds 178-1025 in SEQ 1D 7) PCR amplified and purified, and the purified fragment was labelled32P using Random Prime DNA Labelling Kit (set izgotovlyayuschego solution, having the same composition as the solution to prehybridization. The resulting filter was washed 3 times in 2 x SSC/ 0,1% SDS solution at room temperature for 5 minutes and then 1 time in a solution of 0.1 x SSC/0,1% SDS at 68oC for 1 hour. The filter is then exposed for 16 hours autoradiography at -70oWith using intensifying screen and X-OMATTMAR5 film (Eastman Kodak). the result observed a single band of approximately 10, etc., N. in the case of Hindlll cleavage. Therefore, 10 µg DNA clone HGTl was Hindlll digested and subjected to electrophoresis in 0.8% of agarose gel and the band 10, etc., ad cut out from the gel, purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was subcloned into the cloning vector pUC13 (Pharmacia), which was previously split Hind111. In this case, a competent E. coli strain DH5 (TOOO) was used as a strain-master.

From the thus obtained clones was selected clone containing Hind111 fragment from 10 T. p. H., and was marked pHGT1.

Restriction map of phage clone HGT1 shown in Fig.11.

Example 25. Determination of the nucleotide sequence of chromosome clone pHGT1 TPO man

Cultivation clone pHGTl and purification of plasmid DNA was performed mainly accepted in 50 ml of LB medium, containing 50 μg/ml ampicillin. The resulting cells were collected by centrifugation and suspended in 4 ml of the above solution TED-lysozyme. Thereto was added 8 ml of 0.2 N NaOH /0.1% of SDS and then 6 ml of 3 M potassium/5M acetate for a thorough suspension of cells. After centrifugation of this suspension obtained supernatant was treated with a mixture of phenol/chloroform (1:1), mixed with an equal volume of isopropanol, and then centrifuged. The precipitate was dissolved in THE solution and were treated with RNase and then with a mixture of phenol/chloroform (1:1) followed by precipitation with ethanol. The precipitate was again dissolved in THE solution, to which was then added NaCl and polyethylene glycol 3000 to their final concentrations 0,63 M and 7.5%, respectively. After centrifugation the precipitate was dissolved in THE solution and precipitated with ethanol. In this way received approximately 300 μg of plasmid DNA G1.

Using Taq Dye DeoxyTMTerminater Cycle Sequening Kit (set, manufactured Applied Biosystems) sensitive plasmid DNA was introduced into the DNA sequencing machine A (Applied Biosystems) to determine the nucleotide sequence of the protein coding region predicted from the nucleotide sequence of the cDNA. Certain still is the list of sequences (SEQ 1D 8). In this case, the oligonucleotides used in example 22 for nucleotide analysis, cDNA, and synthetic oligonucleotides based on the internal sequence obtained by analysis of the sequence used as primers in the determination of the nucleotide sequence.

In the result it was found that the chromosomal DNA, plasmid carried by clone G1, contains the complete coding region amino acid sequence derived from SEQ 1D 6, and the nucleotide sequence of the coding region coincided fully with SEQ 1D 6. In addition, the area corresponding to codereuse amino acids to exon contained intron 4, having a length 231 p. N., 286 p. N., 1932 p. N. and 236 p. N. in this order from the 5'-side (Fig. 11). Also, this nucleotide sequence differed from the nucleotide sequence of the cDNA (SEQ 1D 7), with 3 positions, the same as were described in example 22 (miscellaneous provisions between SEQ 1D 6 and 7). I.e., A (position 84) And (position 740) and G (position 1198) in SEQ 1D 7, respectively, T and a in SEQ 1D 8. Thus, it was found that the nucleotide sequence of clone G1 cDNA TRO person, obtained in example 21, differs from the nucleotide sequence of the activities clone pHFT1 cDNA sequence of chromosomal DNA, determined nucleotide sequence of the other 4 clones among the 5 clones of chromosomal DNA, independently selected by this screening. The sequence analysis was performed by direct method of determining the nucleotide sequence using DNA samples of phage obtained according to the method of lysis in the cups described in Molecular Cloning. Sequencing of each clone was performed using DNA sequencing machine A (Applied Biosystems) using the primers used in example 22, which were synthesized on the basis of such parts of the sequence that it was possible to analyze the above 3 positions of mutations in the nucleotide sequence. Used Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems). It was found that the nucleotide sequences of all 4 clones were identical to SEQ 1D 6. Position 84 and 740 corresponding to SEQ 1D 7 have been replaced by s and T, respectively, in these 4 clones. However, the situation 1198 was G 2 clones and in the other two clones. In other words, it was found that there are two types of nucleotide sequences inherent to the source of chromosomal DNA. Currently, it is unclear if such differences in nucleotide sequences of homologous chromosomes or from multiple genes. Ky racial difference, because poly (A)+RNA purchased from Clontech, is of Caucasian origin, whereas the chromosomal DNA is of Japanese origin.

Vector pH T1, carried by the E. coli strain DH5 was deposited by the authors of the present invention 24 March 1994 FERM BP-4616 at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 26. The expression of chromosomal DNA TRO person in COS 1 cells and confirmation of TPO activity

Plasmid clone G1 received by sublimemovies, contained only 4 sequence recognition EcoRl, 3 in the insertion part and 1 in the vector. Analysis of the nucleotide sequence revealed that the entire encoding protein TRO person area was included in the DNA fragment of about 4.3, etc. ad, which was inserted between the sequence of recognition EcoRl, nearest to the 5'-side in the insertion and sequence recognition EcoRl in the vector. Therefore, this fragment is ligated with the processed EcoRl expressing vector pEF18S and got 4 expressing TRO person plasmids 1 - 4 (see Fig.11). Having these plasmid DNA, we conducted an experiment on their expression. Purification of plasmid DNA was performed basically according to the method described in Molecular Cloning (Sambrook et al., Cold S thus clones pFG 1-4 were performed according to the method of example 11. I.e., transfection was performed with 10 μg of plasmid DNA DEAE-dextranomer the method comprising treatment with chloroquine. Transfetsirovannyh COS 1 cells were incubated 3 days at 37oAnd then harvested culture supernatant.

Cultural supernatant extensive were dialyzed against culture medium IMDM and evaluated in the test system rat CFU-MK. TPO activity was found in a dose-dependent form in the supernatant of COS 1 cells, in which expressively each of the four clones (pEFHGTE 1 - 4). In contrast, TPO activity was not detected in the supernatant of COS 1 cells, in which expressives only plasmid pEF18S. Typical results with pEFHGTE 1 shown in Fig.12A. Similar data were obtained in M-e test system. Supernatant of COS 1 cells, in which expressively each of the clones (pEFHGTE 1 - 4), significantly increased the growth of cells M-e dependent on dose. Representative data from pEFHGTE 1 shown in Fig.12.

These results showed that the plasmid clones EFGE 1 - 4 are functional chromosomal DNA TRO person.

Example 27. Obtaining DNA deletion type SRW person and her expression and confirmation of TPO activity

The results in example Inoi thirds. Therefore, to further assess the biologically active parts were experimenting with its deletion derivatives. In this example, deletion derivatives TRO, remote 29, 40, 60, or 68 amino acids from carboxyl-terminal end of the protein TRO (amino acids 1-231) encoded pHT1-231, were investigated on their ability to influence the biological activity of TPO in vitro. The shortest derivative (Amino acids 1-163) still contained the amino acid sequence corresponding to the TR2/3 TRO plasma of rats described in example 2. Deletion plasmids was obtained by PCR using DNA of plasmid clone RT-231, obtained in example 18 as a matrix and the synthesized oligonucleotides as primers. Sequences of primers used for PCR were the following.

hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ 1D 56) (obtained by adding a sequence of recognition EcoRl to the sequence of positions 1-21 in SEQ 1D 4 is identical to the sequence described in Fig.9).

hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTG GTG GGT-3' (SEQ 1D 57) (antisense primer corresponding to the sequence positions 555-576 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and consequently what now remains,

h-4: 5'-TTT GCG GCC GCT CAT TAC AGT GTG AGG ACT AGA GAG GTT CTG-3' (SEQ 1D 58) (antisense primer corresponding to the sequence positions 576-600 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-171),

hTPO-30: 5'-TTT GCG GCC GCT CAT TAT CTG GCT GAG GCA GTG AAG TTT GTC-3' (SEQ 1D 59) (antisense primer corresponding to the sequence positions 636-660 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-191), and

. hTPO-2: 5'-TTT GCG GCC GCT CAT TAC AGA CCA GGA ATC TTG GCT CTG AAT-3' (SEQ 1D 60) (antisense primer corresponding to the sequence positions 696-720 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-211).

Using 1 μg of plasmid DNA of clone pHT1-231, obtained in example 18, as a matrix, 10 μm of each of the synthesized h-5 (5'-side) and h-2, -3, -4 and S (3'-crocodile R in a volume of 100 μl using R system 9600 GeneAmpTM(PERKIN-ELMER) with the repetition of 20 cycles each of which consisted of denaturation at 95oC for 1 min, hybridization at 65oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes. The target bandwidth obtained by each R, were digested with restrictase EcoRl and Notl and the resulting cleavage product was subjected to electrophoresis in 1% agarose gel (F BioProducts) to highlight the main DNA fragment with the expected size of the amplified each R. Underlined DNA fragment was purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and then was subcloned into expressing vector pEF18S, previously cleaved with the same restrictases. In this case, a Competent E. coli strain DH5, made TOOO, was used as a strain-owner. From the obtained transformants, each of which came from its PCR were selected 4-5 clones containing the insertion of the expected size, to obtain samples of plasmid DNA, mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In some of these operations were obtained samples of plasmid DNA from the deletion derivative, codereuse slotnik residues (pHT1-171 1-4), deletion derivative encoding the position of the amino acid residues 1-191 (pHT1-191 1-4) and deletion derivative encoding the position of the amino acid residues 1-211 (pHT1-211 1-4). The overall length of the amplified regions of each plasmid DNA were subjected to analysis of nucleotide sequence to find its complete identity with the nucleotide sequence shown in SEQ 1D 4.

Transfection in COS 1 cells thus obtained clones were performed according to the method of example 11. I.e., transfection was performed with 10 μg of each of the samples of plasmid DNA by DEAE-dextranomer the method comprising treatment with chloroquine. Transfetsirovannyh COS 1 cells were cultured for 3 days and then harvested culture supernatant.

These cultural supernatant extensive were dialyzed against culture medium IDM and evaluated in test/system with rat CFU-MK. TPO activity was detected (dose-dependent) in the supernatant of COS 1 cells, in which expressives each of pHT1-211, pHT1-191, pHT1-171 or pHT1-163. Representative data from pHT1-211 1, RNT-191 1 and pHT1-171 2 shown in Fig. 13A and pHT1-163 2 in Fig.13. Similar results were obtained in M-e-test the system. Supernatant of COS 1 cells, in which aktivnye data from pET1-211 1, pET1-191 1, pHT1-171 2 or pHT1-163 2 shown in Fig.14.

These results showed that SRAW man still retains its biological activity in vitro even after deletions his carboxyl-terminal half for Ser (position 163), and gave a strong basis for assuming that the biologically active part of the TRO person located within the amino-terminal half, ending at Ser (position 163).

Example 28. Obtaining TRO person with a deletion at the C-end and its expression and activity in COS 1 cells is

For analysis of the district, necessary for protein TRO man showed his activity, C-terminal amino acids was deleterule of deletion derivatives obtained in example 27, and felt TPO activity, downregulation of derivatives obtained in COS 1 cells. Using plasmid clone pEF18S-HL34 obtained in example 16 was received expressing plasmids by deletions of the nucleotide sequences encoding the C-terminal amino acid residues, starting with serine at position 163. The design of these deletion plasmids was performed using PCR. In this process we used the following primers:

. hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ 1D 61) (obtained by adding after the while example 18),

. hTPO-150: 5'-TTT GCG GCC GCT CAT TAG AGG GTG GAC CCT CCT ACA AGC AT-3' (SEQ 1D 62) (antisense primer corresponding to the sequence positions 514-537 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl for use in obtaining deletion mutant encoding the position of the amino acid residues 1-150),

. hTPO-151: 5'-TTT GCG GCC GCT CAT TAG CAG AGG GTG GAC CCT CCT ACA A-3' (SEQ 1D 63) (antisense primer corresponding to the sequence positions 518-540 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-151),

.h-153: 5'-TTT GCG GCC GCT CAT TAC CTG ACG CAG AGG GTG GAC CC-3' (SEQ 1D 64) (antisense primer corresponding to the sequence positions 526-546 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-153),

.hTPO-154: 5'-TTT GCG GCC GCT CAT TAC CGC CTG ACG CAG AGG GTG GA-3' (SEQ 1D 65) (antisense primer corresponding to the sequence positions 529-549 in SEQ 1D 4 obtained by adding two termination codons situation amino acid residues 1-154),

.h-155: 5'-TTT GCG GCC GCT CAT TAG GCC CGC CTG ACG CAG AGG GT-3' (SEQ 1D 66) (antisense primer corresponding to the sequence positions 532-552 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-155),

.hTPO-156: 5'-TTT GCG GCC GCT CAT TAT GGG GCC CGC CTG ACG CAG AG-3' (SEQ 1D 67) (antisense primer corresponding to the sequence positions 535-555 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl, for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-156), and

.hTPO-157: 5'-TTT GCG GCC GCT CAT TAG GGT GGG GCC CGC CTG ACG CA-3' (SEQ 1D 68) (antisense primer corresponding to the sequence positions 538-558 in SEQ 1D 4 obtained by adding two termination codons TAA and TDA and sequence recognition Notl for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-157).

PCR was performed using 1 μg of plasmid DNA of clone pEF18S-HL34 obtained in example 16, as a matrix, and 10 μm of each of the synthesized so oligonucleotides (hTPO-5 for 5 is for GeneAmp PCRTMwith AmpliTaqTMDNA polymerase (manufactured by Takara Shuzo) was performed PCR in a 100 μl volume, using a PCR system 9600 GeneAmpTM(PERKIN-ELMER) and repeat 20 cycles, each of which consisted of denaturation at 95oC for 1 min, hybridization at 66oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes. Target strip from each PCR were digested with restrictase EcoRl and Notl and the resulting cleavage product was subjected to electrophoresis in 1% agarose gel (FMC BioProducts) to highlight the main DNA fragment with the expected size for each PCR. Underlined DNA fragment was purified using the kit for purification of DNA Rger-A-Gene (Bio-Rad) and then was subcloned into expressing vector pEF18S, pre-processed by the same restrictases. In this case, a Competent strain of E-coli DH5 (TOOO) was used as a strain-owner. From the obtained transformants originating from each R, selected 3-5 clones containing the insertion of the expected size, to obtain samples of plasmid DNA essentially in accordance with the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Through a series Slotnik residues (pHT1-150 21, 22 and 25), deletion derivative, encodes the position of the amino acid residues 1-151 (pHT1-151 16, 17 and 18), deletion derivative, encodes the position of the amino acid residues 1-153 (RNT-153 1-5), deletion derivative, encodes the position of the amino acid residues 1-155 (pHT1-155 1-5), deletion derivative, encodes the position of the amino acid residues 1-156 (pHT1-156 1-5), and deletion derivative, encodes the position of the amino acid residues 1-157 (pHT1-157 1-5).

Of these purified sample of plasmid DNA sequence RNT-150 21, 22 and 25 and pHT1-151 16, 17 and 18 were checked by DNA sequencing machine A (Applied Biosystems) using the Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems), confirming that the expected sequence of cDNA TRO had no replacements in all nucleotide sequences.

Transfection of COS 1 cells, each of the thus obtained clones was carried out according to the method of example 11. I.e., transfection was performed with 10 μg of each of the samples of plasmid DNA DEAE-dextranomer way capable of handling chloroquine, and the culture supernatant was recovered after 3 days of cultivation. The obtained culture supernatant were dialyzed against culture medium ID described earlier, and assess svirirojana relevant clones, encoding the C-terminal deletion derivatives, consisting of the positions of the amino acids 1-151, the provisions of amino acids 1-153, the provisions of amino acids 1-154, the provisions of amino acids 1-155, the provisions of amino acids 1-156 or provisions of amino acids 1-157. All these derivatives contain a cysteine residue at position 151. However, TPO activity were not detected in the culture supernatant of COS 1 cells transfected clone coding for deletion derived C-terminal side, consisting of the positions of the amino acids 1-150, in which the cysteine residue at position 151 was also deleterows.

Example 29. Obtaining TRO person with a deletion of the N-end and its expression and activity in COS 1 cells is

For analysis of the district, necessary for protein TRO showed its biological activity, deletion derivative with a deletion of amino acids at N-end, obtained in example 28, was deleterule next, and the activity of TPO in the received deletion derivatives were determined. Using plasmid clone EF18S-HL34 obtained in example 16, and the plasmid clone pHT1-163, obtained in example 27, expressing plasmid was obtained by deletion of the nucleotide sequences encoding the N-terminal amino acid residues, going after C the activity of primers, made for use in PCR are as follows:

h-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID 69) (obtained by adding a sequence of recognition EcoRl to the sequence of positions 1 to 21 in SEQ ID 4, the same sequence shown in example 18),

hTPO-3: 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTC AGG TFT CC-3' (SEQ ID 70) (antisense primer corresponding to the sequence positions 757-780 in SEQ ID 4, obtained by adding two termination codons TAA and TDA and sequence recognition Notl; the same sequence synthesized for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-231),

. hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG AGA GCT GTG GTG GGT-3' (SEQ ID 71) (antisense primer corresponding to the sequence positions 555-576 in SEQ ID 4 obtained for use in obtaining a deletion derivative encoding the position of the amino acid residues 1-163),

.hTPO-13: 5'-AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG TG-3' (SEQ ID 72) (position 124-173 in SEQ ID 4, obtained for use in obtaining a derivative in which deleterows amino acid residues of positions 1-12),

. hTPO-13R: 5'-CAT GGG AGT CAC GAA GCA GTT TAC TGG ACA GCG TTA GCC TTG CAG TTA G-3' (SEQ ID 73) (antisense primer corresponding placentas is designed amino acid residues of positions 1-12),

hTPO-7: 5'-TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT C-3' (SEQ ID No. 74) (position 106-154 in SEQ IG 4, obtained for use in obtaining a derivative in which deleterows amino acid residues of positions 1-6),

.hTPO-7R: 5'-TTT ACT GAG GAC TCG GAG GTC ACA GGA CAG CGT TAG CCT TGC AGT TAG -3' (SEQ ID 75) (antisense primer corresponding to the sequence positions 64-87 and 106-129 in SEQ ID 4, prepared for use in obtaining a derivative in which deleterows amino acid residues of positions 1-6),

. hTPO-8: 5'-GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC A-3' (SEQ ID 76) (position 109-157 in SEQ ID 4, obtained for use in obtaining a derivative in which deleterows amino acid residues of positions 1 to 7),

.hTPO-8R: 5'-CAG TTT ACT GAG GAC TCG GAG GTC GGA CAG CGT TAG CCT TGC AGT TAG-3' (SEQ ID 77) (antisense primer corresponding to the sequence positions 64-87 and 109-132 in SEQ ID 4, obtained for use in obtaining a derivative in which deleterows amino acid residues of positions 1-7).

(1) derivatization (RT-231), in which deleterows amino acid residues of positions 1-12

R was performed using a 1.4 μg of plasmid DNA of clone pEF18S-HL34 obtained in example 18, as a matrix, and 5 μm of each of the synthesized so oligonucleotides (hTPO-13 and h is for GeneAmp PCRTMwith AmpliTaqTMDNA polymerase (Takara Shuzo) was performed PCR in a 100 μl volume using a PCR system 9600 GeneAmpTM(PERKIN-ELMER) and, after 5 minutes of denaturation at 95oWith repeated generally 30 cycles, each of which consisted of denaturation at 95oC for 1 min, hybridization at 65oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes. Each of the PCR products were subjected to electrophoresis in 1.2% agarose gel (izgotovlena FMC BioProducts) to highlight the main product of the DNA with the expected size, which was then purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was dissolved in 15 μl of TE buffer. After this was performed a second PCR using 1 µl of each of the thus prepared solutions as a matrix.

The second PCR was performed using 5 μm of each of the synthesized primers (h-5 and hTPO-3). Using a set of reagents for PCR, GeneAmpTMwith AmpliTaqTMDNA polymerase (Takara Shuzo) PCR was performed in a 100 μl volume using PCR system 9600 Gene-AmpTM(PERKIN-ELMER), and after 5 minutes of denaturation at 95oWith repeated a total of 30 cycles, each of which consisted of denaturation at 95othe subsequent final incubation at 72oC for 1 minute. Each of the products R were subjected to electrophoresis in 1.2% agarose gel (FMC BioProducts) to highlight the main DNA fragment with the expected size, which was then purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was dissolved in 15 μl of TE buffer. After splitting restrictase EcoRl and Notl the resulting solution was subjected to extraction with the same volume of a mixture of phenol/chloroform followed by ethanol precipitation. After centrifugation the precipitate was dissolved in 15 μl of TE buffer and then was subcloned into expressing vector pEF18S, pre-processed by the same restrictases. Competent E. coli DH5 (TOOO) was used as a strain-owner. From the obtained transformants were selected 45 clones containing the insertion of the expected size for obtaining samples of plasmid DNA mainly in accordance with the method described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989). In this way it was possible to obtain plasmid DNA deletion derivative (RT-231), encoding a protein molecule, in which amino acid residues of positions 1-12 were deleterows from the original amino acid sequence 1-231. When conducting R these 45 clones using primers hTPO-5 and hTPO-3 insertions, the La tested in a DNA-sequencing machine A (Applied Biosystems) using the Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems) to obtain the resulting clone RT-231 cDNA sequence, which wanted to construct, and deletions, which was expected, without substitutions and other changes to the entire nucleotide sequence.

(2) derivatization (RNT-163), in which deleterows amino acid residues of positions 1-6

In this case, the amino acid position 163 was used as C-end to obtain deletion derivatives, although in the preceding stage used the amino acid position 231 as of late protein TRO to obtain a deletion derivative. It was found that TPO activity could be expressed, even when further DeleteMovie C-terminal amino acids. For this reason, the position of 163 amino acids used as the end in the next stage (3). PCR was performed using a 1.4 μg of plasmid DNA of clone pHT1-163, obtained in example 27, as a matrix, and 5 μm of each of the synthesized oligonucleotides (hTPO-7 and hTPO-S) in one combination and hTPO-5 and hTPO-7R in other combination) as primers. Using a set of reagents for PCR GeneAmpTMwith AmpliTaqTMDNA polymerase (manufactured by Takara Shuzo) PCR was performed in a 100 µl volume using the each of which consisted of denaturation at 95oC for 1 minute, renaturation at 65oC for 1 min, and synthesis at 72oC for 1 min, with a final incubation at 72oC for 7 minutes. Each of the PCR products were subjected to electrophoresis in 1.2% agarose gel (izgotovlena FMC BioProducts) to highlight the main DNA fragment with the expected size, which was then purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was dissolved in 15 μl of TE buffer. After this was performed a second PCR using 1 µl of each of the thus obtained solutions as a matrix.

Second R was performed using 5 μm of each of the synthesized primers (hTPO-5 and hTPO-S). Using a set of reagents for PCR GeneAmpTMwith AmpliTaqTMDNA polymerase (Takara Shuzo) PCR reaction was performed in a 100 μl volume using PCR system 9600 GeneAmpTM(PERKIN-ELMER) and, after 5 minutes of denaturation at 95oWith repeated generally 30 cycles, each of which consisted of denaturation at 95oC for 1 minute, renaturation at 60oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes. Each of the PCR products were subjected to electrophoresis in 1.2% agarose gel (Ali with the use of the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was dissolved in 15 μl of TE buffer. After splitting restrictase EcoRl and Notl the resulting solution was subjected to extraction with the same volume of a mixture of phenol/chloroform followed by ethanol precipitation. After centrifugation the precipitate was dissolved in 15 μl of TE buffer and then was subcloned into expressing vector pEF18S, which was pre-treated with the same restrictases. In this case, a Competent E. coli strain DH5 (TOOO) was used as a strain-owner. From the obtained transformants were selected 30 clones containing the insertion of the expected size, to obtain samples of plasmid DNA mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Sprining Harbor Laboratory Press, 1989). In this way it was possible to obtain plasmid DNA deletion derivative (RNT-163), encoding a protein molecule, in which amino acid residues of positions 1-6 were deleterows from the initial sequence of amino acid residues 1-163 in SEQ ID 4. When conducting these clones PCR using primers hTPO-5 and hTPO-S insertions that have approximately the expected size was confirmed in all clones. One sequence of 3 clones were verified using DNA sequencing machine A (Applied Biosystems) using Taq Dey DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems) with recip, the deletions, which was expected, without replacement, and any other changes in the entire nucleotide sequence.

(3) derivatization (RNT-163), in which deleterows amino acid residues of positions 1-7

Derived (RNT-163) with a deletion of the amino acid residues of positions 1 to 7 were prepared in much the same way as in the above case, when deriving (RNT-163) with a deletion of the amino acid residues of positions 1-6. PCR was performed using a 1.4 μg of plasmid DNA of clone pHT1-163, obtained in example 27, as a matrix, and 5 μm of each of the synthesized oligonucleotides (hTPO-8 and hTPO-S in one hand and h-5 and hTPO-8R in other combination) as primers. Using a set of reagents for PCR GeneAmpTMwith AmpliTaqTMDNA polymerase (Takara Shuzo) the PCR reaction was performed in a 100 μl volume using R system 9600 GeneAmpTM(PERKIN-ELMER) and, after 5 minutes of denaturation at 95oWith repeated generally 30 cycles, each of which consisted of denaturation at 95oC for 1 minute, renaturation at 65oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes. Each of the PCR products were subjected to electrophoresis in 1, is imali with the use of the kit for purification of DNA Prep-A-Depe (Bio-Rad) and was dissolved in 15 μl of TE buffer. After this was performed a second PCR using 1 µl of each of the obtained solutions as a matrix.

Second R was performed using 5 μm of each of the synthesized primers (hTPO-5 and hTPO-S). Using a set of reagents for PCR GeneAmpTMwith AnipliTaqTMDNA polymerase (manufactured by Takara Shuzo) was performed PCR in a 100 μl volume using PCR system 9600 GeneAmpTM(PERKING-ELMER) and, after 5 minutes of denaturation at 95oWith repeated generally 30 cycles, each of which consisted of denaturation at 95oC for 1 minute, renaturation at 60oC for 1 min, and synthesis at 72oC for 1 min, with a subsequent final incubation for 7 minutes. Each of the PCR products were subjected to electrophoresis in 1.2% agarose gel (FMC Bio-Products) to highlight the main DNA fragment with the expected size, which was then purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad) and was dissolved in 15 μl of TE buffer. After splitting restrictase EcoRl and Notl the resulting solution was subjected to extraction with the same volume of a mixture of phenol/chloroform and subsequent precipitation with ethanol. After centrifugation the precipitate was dissolved in 15 μl of TE buffer and then was subcloned into expressing the m E. coli DH5 (TOYOBO)

used as strain-owner. From the obtained transformants were selected 30 clones containing the insertion of the expected size for obtaining samples of plasmid DNA mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way it was possible to obtain plasmid DNA deletion derivative (RNT-163), encoding a protein molecule, in which amino acid residues of positions 1-7 were deleterows from the original amino acid residues of the provisions 1-163 in SEQ ID No 4. When conducting these clones PCR using primers hTPO-5 and hTPO-S insertions that have approximately the expected size was confirmed in all clones. Among them, the sequence of the 3 clones was verified by DNA-sequencing machine A (Applied Biosystems) using the Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems) to obtain the 2 clones, RT-163 33 and 48, each of which has a cDNA sequence TRO, which was designed and deletions, which was expected, without substitutions or similar changes in the entire nucleotide sequence.

(4) the Expression of deletion derivative in COS 1 cells and confirmation of TPO activity

Transfection of COS 1 cells, each of the thus obtained deletion clones PR is the tax DEAE-dextranomer way capable of handling chloroquine, and cultural supernatant was extracted after 3 days of cultivation. Thus obtained culture supernatant were dialyzed thoroughly against culture medium IMDM described above, and were evaluated using the test system in M-e.

The result is a weak, but significant, TPO activity was detected in the culture supernatant of COS 1 cells transfected clone coding for deletion derivative, consisting of amino acids provisions 7-163. However, TPO activity was not detected in the culture supernatant of COS 1 cells transfected clone coding for deletion derivative, consisting of amino acids provisions 8-163 or amino acids provisions 13-231.

Example 30. Obtaining the plasmid cDNA (pHTP1) SRW full length human

Expressing the vector with all encode the amino acid regions of cDNA TRO person alleged in SEQ ID 6, was constructed for expression in mammalian cells.

R was carried out as follows to obtain a DNA fragment covering all coding regions of cDNA TRO person.

The nucleotide sequences of used primers were as follows:

. hTPO-1: 5' TTG TGA CCT C is ejstvujuschij sequence positions 745-765 in SEQ ID No 6);

. hTPO-P: 5'-TGC GTT TCC TGA TGC TTG TAG-3' (SEQ ID No 80) (position 503-523 in SEQ ID 6);

. hTPO-KO: 5'-GAG AGA GCG GCC GCT TAC CCT TCC TGA GAC AGA TT-3' (SEQ ID No 81) (sequence obtained by adding a sequence of recognition Notl and sequence GAGAGA (SEQ ID No 82) to the antisense sequence corresponding to the sequence positions 1066-1086 in SEQ ID No 6).

The first PCR was carried out using 300 ng of clone pEF18S-HL34 obtained in example 16, as a matrix. Using 0.5 µm of each primer hTPO-1 to SA I E Vent RTMDNA polymerase (manufactured by New England Biolabs) was performed R (repetition in General, 30 cycles each of which consisted of incubation at 96oC for 1 minute incubation at 62oC for 1 minute incubation at 72oC for 1 min, with a subsequent final incubation at 72oC for 7 minutes). The composition of the reaction solution at final concentrations were as follows: 10 mm KS1, 10 mm (NH4)2SO4, 20 mm Tris-HC1 (pH 8.8), 2 mm MgS4, 0.1% Triton X-100 and 200 μm dNTP mixture.

1 μg of a commercially available preparation of poly (A)+RNA obtained from normal human liver (produced by Clontech), was heated at 70oC for 10 minutes, rapidly cooled on ice and then mixed with 10 MSY TO-(manufactured by LIFE TECHNOLOGIES) followed by incubation for 1 hour at 37oFor carrying out the synthesis of cDNA. Then spent the second PCR (repetition in General 30 cycles, each cycle consisted of incubations at 96o1 min, at 58o1 min and at 72oWith 1 min) using 1/20 of the volume of the reaction solution of the synthesized cDNA as the template and 2.5 μm each of primers hTPO-P and hTPO-KO and 2.5 units AmpliTaqTMDNA polymerase (produced by Takara Shuzo).

Each of the obtained solutions of the first and second R were subjected to electrophoresis in 1% agarose gel to highlight the corresponding basic DNA fragments of the expected size, which are then purified using the kit for purification of DNA Prep-A-Gene (manufactured by Bio-Rad). Then spent a third PCR using 1/20 of the volume of each of the thus obtained solutions as a matrix. This reaction (heating at 96o2 min, followed by repetition of only 3 cycles, each of which consisted of incubations at 96o2 min and 72o2 min, followed by incubation at 72oC for 7 minutes) using 1 unit of Vent RTMDNA polymerase (manufactured by New England Biolabs). The resulting reaction solution Pach is which consisted of incubation at 96oWith 1 min at 62o1 min and at 72o1 min, followed by incubation at 72oC for 7 minutes. The resulting reaction solution was extracted with the same volume of saturated water mixture phenol/chloroform followed by precipitation with ethanol (2.5 volumes of ethanol in the presence of 0.3 M sodium acetate and 0.5 ál glycogen produced by Boehringer-Mannheim, extract the DNA. Thus obtained DNA was digested with restrictase BamH1 and Notl and subjected to electrophoresis in 1% agarose gel and the main band with the expected size of the allocated thus, purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad), ligated with the vector pBLUESCRIPT 11 SK+(Stratagene), which was previously split restrictase BamH1 and Notl, and then transformed into a Competent E. coli strain DH5 (TOYOBO). From the obtained colonies were selected 4 clone to obtain samples of plasmid DNA. The sequence of the purified sample of plasmid DNA was verified using DNA sequencing machine A manufactured by Applied Biosystems using Taq Dye DeoxyTMTerminater Cycle Sequening Kit (Applied Biosystems), resulting in the clone, pBLTP with the cDNA sequence TRO designed without substitutions in the nucleotide sequence within the region between the BamH1 and Notl.

high molecular weight, selected thus purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad). In the same way PEF18S-HL34 were treated with restrictase for the purification of a DNA fragment of about 450 p. N. Each of the thus obtained DNA samples were subjected to legirovanie and transformed into Competent E. coli strain DH5 (TOOO). Samples of plasmid DNA were obtained from the resulting colonies to obtain clone, pBLTEN containing the insertion of cDNA TRO person. Thus obtained clone pBLTEN were digested with restrictase EcoR1 and Notl and subjected to electrophoresis in 1% agarose gel, and the DNA fragment of about 1200 p. N., underlined, was purified using the kit for purification of DNA Prep-A-Gene (Bio-Rad), ligated with expressing vector pEF18S, which had been previously cleaved with the same restrictases, and then transformed into Competent strain DH5 E. Li (TOOO). Samples of plasmid DNA were obtained from the obtained colonies were clone, RNT containing the coding region of cDNA of the entire SRW person. Plasmid DNA was obtained from this clone in large quantities and used in further experiments. Plasmid DNA isolation was performed basically according to the method described in Molecular Cloning (Sambrok et al., Cold Spring Harbor Laboratory Press, 1989)man in the cells of the Chinese hamster ovary (Cho)

1 µg of plasmid G1 containing minigun mouse digidrofolyatreduktazy (DHFR), were digested with restrictase EcoR1 and BamH1 and subjected to agarose gel electrophoresis for separation of the fragment (approximately 2.5, etc., ad), containing minigun mouse DHFR. The selected DNA fragment was dissolved in 25 ml of reaction solution consisting of 50 mm Tris-model HC1 (pH 7.5), 7 mm MgCl2, 1 mm 2-mercaptoethanol and 0.2 mm dNTP mixture, was mixed with 2 units of fragment maple and then incubated at room temperature for 30 minutes for the formation of blunt ends at both ends of this DNA fragment. After treatment with a mixture of phenol/chloroform and precipitation with ethanol, the obtained fragment was dissolved in 10 ml of TE solution (10 mm Tris-model HC1 (pH 8.0)/1 mm EDTA). Expressing the vector mammals pEF18S were digested with Sma1 restriction enzyme, was dephosphorylated alkaline phosphatase (manufactured by Takara Shuzo), and then ligated with a DNA fragment containing minigun murine DHFR, using T4 DNA ligase (Takara Shuzo), receiving expressing vector DF202.

Then constructed expressing vector pDEF202 were digested with restrictase EcoR1 and Spel and then subjected to agarose gel electrophoresis for separation of large DNA fragment. cDNA TRO person, which was poetin linearized vector pDEF202 using T4 DNA ligase (Takara Shuzo), getting plasmid F202-h-P1 for the expression of cDNA TRO person. This constructed plasmid contains the beginning of the replication of SV40, 1-- the promoter of the elongation factor of the person and early polyadenylation signal SV40 transcription cDNA TRO person, minigene murine DHFR and start replication pUC18 and gene-lactamase (Ampr).

Example 32. The expression of TPO person in Cho cells

Cho cells (DHFR - strain, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, vol.77, p.4216, 1980) were maintained in minimal basic medium (-MEM(-), with the addition of gipoksantina and thymidine) containing fetal calf serum (FCS), using cups for tissue culture 6 cm in diameter (Falcon). The transformation of Cho cells plasmid pDEF202-hTPO-P1 was performed by calcium-phosphate method (CellPhect, Pharmacia), as described below. 10 μg of plasmid pDEF202-hTPO-P1 obtained in example 31, was mixed with 120 ml of buffer a and 120 ml of N2Oh and the mixture was incubated at room temperature for 10 minutes. This solution is then mixed with 120 ml of buffer and allowing it to stand at room temperature for another 30 minutes. This solution mixture of DNA was then added to the culture and incubated for b hours in the incubator with CO2. After 6 hours, the culture was washed twice in serum-free a-MEM(-), obrabatyvali medium (-MEM (-) with the addition of gipoksantina and thymidine), containing 10% cialisbuynow FS. After 2 days of cultivation, the cells were selected on selective medium (-MEM ( -)) containing 10% cialisbuynow FCS. For selection of transformed cells with DHFR - positive phenotype of these cells was treated with a solution of tipsin/EDTA and resuspendable in the electoral environment. Cells in the Cup for tissue culture (6 cm) was divided into 5 cups (10 cm) or 12 to 24-hole culture of tablets and then cultured in this electoral environment. The culture medium was replaced at intervals of 2 days. Activity SRW person in the culture medium of Cho cells with DHFR-positive phenotype was measured by CFU-MK - test, M-e-test or VA/P3 test. Cells secreting TRO person in the culture medium, were treated with 25 nm methotrexate for isolation of cell clones that produce high amount of waste man.

In this case, the transfection of Cho cells can also be performed by holding cotransfection RMS cells with plasmids rntr and G1.

SNO strain (SNO - DUKXB11), transfetsirovannyh the plasmid pDEF202-hTPO-P1, was deposited by the author of this invention on January 31, 1995, under FERM BP-4988 National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 33. Consciouses vector mammals pBMCGSneo were digested with restrictase Xhol and Notl and subjected to agarose gel electrophoresis for the separation of part of the vector DNA. This fragment DNA and cDNA TRO person (P1 clone), which was obtained by cleavage of the plasmid pBLTEN containing cDNA TRO person, restrictase Xhol and Notl ligated using T4 DNA ligase, receiving targeted expressing vector, pBMCGSneo-hTPO-P1. This expressing plasmid contains the early promoter of cytomegalovirus, part derived from gene-rabbit globin intron and polyadenylation area for transcription cDNA TRO human gene-globin man, 69% virus gene 1 bovine papillomavirus, promoter timedancing and polyadenylation district timedancing, gene phosphotransferase 1 (resistant to neomycin gene) and the beginning (origin) replication R322 and gene-lactamase (AMRr) and cDNA TRO person was Legerova site, located in the direction 5'-3' from the promoter of cytomegalovirus.

Example 34. Expression in X 63.6.5.3. cells

X 63.6.5.3 cells were cultured in basic medium, Dulbecco (DME) containing 10% fetal bovine serum. Transfection Of X. 63.6.5.3. cells plasmid BMCGSneo-hTPO was performed by the method of electroporation, as described below.

20 μg of plasmid BMCGSneo-hTPO-P1 obtained in example 33 was added to 750 μl of DME medium containing 107cells, and the mixture was placed in a cuvette for electroporation, the ima is Orazio (WITH 600, produced VTH) to conduct gene transfer under conditions of 380 V, 25 MF and 24 angstroms. After gene transfer cell was again allowed to stand for 15 minutes at 4oC. Then cells were washed once DEM containing 10% fetal bovine serum. Transfetsirovannyh cells suspended in 50 ml of DEM with 10 FCS, poured into wells of 96-well culture tablet and then were cultured for 2 days in CO2-incubator. After 2 days of cultivation, the culture medium was replaced DEM containing 10% FCS and 1 mg/ml G418 (produced by GIBCO) and change the medium was then every 3 days. Activity SRW person in the culture medium of the transformants was measured tests with CFU-MK, M-e or BA/F3. Transformants secreting TRO person in the culture medium, cloned twice the limiting dilution to establish producing TRO human cell lines.

Example 35. Large-scale expression TRO person in COS 1 cells is

Transfection of COS 1 cells was performed according to DEAE-dextranomer the method comprising treatment with chloroquine, as described in example 11. COS 1 cells (ATSC CRL 1650) were cultured in IMDM containing 10% (vol./about.) FCS using coated collagen 175 cm2cultural the genome culturalia flask 25 ml solution of collagen (Cellmatrix type 1-C, machines are manufactured by Iwaki), the concentration of which was brought to 0.3 mg/ml using 1 mm model HC1 was added to each 175 cm2culture flask, a solution of collagen was removed after 1 hour of standing at room temperature, and then the thus treated flask was washed 1 time in 20-50 ml of PBS. Transfection was performed by mixing 20 ml IMDM containing 250 µg/ml DEAE-dextran (Pharmacia), 60 μm chloroquine (Sigma) and 10% (vol./about.) Nu-serum (Collaborative) with MTR (40 mg), which was dissolved in 500 μl HBS, adding the resulting mixture to COS 1 cells, described above, is contained in a single 175 cm2culture flask, which were washed once with IMDM directly before transfection. Then these cells were cultured for 3 hours at 37oWith 5% CO2-incubator. Thereafter, the culture supernatant was removed by aspiration and cells were washed once with IMDM, was mixed in a 50 ml serum-free medium and then cultured for 5 days at 36oWith 5% CO2-incubator for extracting the culture supernatant. In one operation used 100-260 culture flasks and extracted 5-13 l of culture supernatant. Serum-free medium were prepared by adding to IMDM 5 μg/ml insulin (produced by Sigma), 5 µg/ml transfer highly purified bovine albumin, produced Nichirei).

Example 36. Purification of full-TRO person (obtained from expressing plasmids rntr, P1 clone) expressed in COS 1 cells, and its biological activity

Here are examples of activity and purification of full-TRO person (obtained from expressing plasmids pHTP1), expressed in COS1 cells. Partially purified product was received first, to test its increased platelet activity and then spent cleaning, getting a product of high purity. To measure TPO activity in subsequent stages were mostly M-e the test system. Similarly TPO activity was determined in the test system rat CFU-MK. When conducting these tests to each sample was added to serum human albumin to a final concentration of 0.02 to 0.05%.

First, COS 1 cells, transfetsirovannyh the plasmid pHTP1 according to the method Ohashi and Sudo (Hideya Ohashi and Tadashi Sudo, Biosci. Biotech. Biochem. , 58 (4), 758-759, 1994) were cultured for 5 days at 37oWith 5% CO2with the use of serum-free culture medium 1MDM containing 0.2 g BSA, 5 mg bovine insulin, 5 mg of human transferrin, 0.02 mm of monoethanolamine and 25 nm of sodium Selenite in 1000 ml receiving the result of the ski enzymes p-APMSF Pefabloc SC hydrochloride (4-(2-amino-ethyl)-benzosulfimide, made Merk cat. 24839) to a final concentration of approximately 1 mm, followed by filtration with a filter of 0.2 μm to obtain a supernatant. The supernatant was concentrated approximately 10-fold using ultrafiltration devices (Omega Ultrasette, with cut-off nominal mol. weight 8000, manufactured Filtron; or PLGC Pellicon Cassette, clipping mol. weight of 10,000, Millipore). 723 ml of this concentrate (protein concentration to 3.38 mg/ml, total protein 2445 mg, the relative activity 43000 total activity 105100000) was mixed with 1.6 moles per 1000 ml of ammonium sulfate (288 g in General), getting 804 ml of a solution containing 1.5 M ammonium sulfate (final concentration). Then the thus obtained solution was applied at a flow rate of 10 ml/min, a column of Macro-Prep Methyl HIC (diameter 5 cm and height 9 cm) of Bio-Rad, cat. 156-0080), which was pre-equilibrated to 20 mm sodium citrate (buffer) (pH 5,2) containing 1.5 M ammonium sulfate. After loading of the samples was performed elution of 20 mm sodium citrate buffer (pH 5,2) containing 1.25 M ammonium sulfate, receiving passing through the column fraction F1 (2384 ml, protein concentration 0,864 mg/ml, total protein 2061 mg, the relative activity of 6000).

Then eluting the solution was replaced with 20 mm sodium citrate, pH Chennai thus the fraction F2 (1081 ml) Macro-Prep Methyl HIC - the column was applied at flow rate 10 ml/min, column, SP Sepharose Fast Flow (diameter 3 cm and height 10 cm) manufactured by Pharmacia Biotech cat. 17-0729-01), which was pre-equilibrated to 20 mm buffer of sodium citrate (pH of 5.8). After loading of the samples was performed elution of 20 mm sodium citrate buffer (pH 5,6) containing 110 mm NaCl, collecting the fraction F1 (2262 ml, protein concentration 0,270 mg/ml, total protein 610 mg, the relative activity of 30,000). Then eluting the solution was replaced with 20 mm sodium citrate (pH of 5.4) containing 400 mm NaCl, collecting the fraction F2 (856 ml, protein concentration 0,189 mg/ml, total protein 162 mg, the relative activity 300000). Then eluting the solution was replaced with 20 mm sodium citrate buffer (pH 5,2), containing 1000 mm NaCl, collecting elyuirovaniya fraction F3 (370 ml, protein concentration 0,034 mg/ml, total protein 12.6 mg, the relative activity 150000).

The main TRO-active fraction F2 (845 ml) stage column of SP Sepharose Fast Flow was mixed with approximately 10% at a final concentration of 1-propanol and was applied at flow rate 3 ml/min to the column LA-WGA (diameter 2 cm, height 14 cm, Honen cat. WG-007), pre-equilibrated to 20 mm sodium citrate buffer (pH 5,4) containing 400 mm NaCl. After loading the column elution was performed with a mixture of buffer 20 mm sodium citrate (who 1,15 mg, relative activity 17220). Then eluting the solution was replaced with 20 mm sodium citrate buffer (pH 6,1) containing 0.4 M GlcNAc and 10% 1-propanol, collecting elyuirovaniya fraction F2 (45 ml, protein concentration 0,0104 mg/ml, total protein 0,470 mg, the relative activity 675000).

The main TRO-active fraction F2 (340 ml) of the operation of the column LA-WGA was mixed with approximately 0,005% in final concentration F and subjected to column chromatography on a YMC-Pack CN-AP (6 mm in diameter and 250 mm height), manufactured UMC, cat. AR-513. With 0.1% F as a developing solvent a and 1-propanol containing 0.05% F, as developing solvent To the sample was applied at a flow rate of 0.6 ml/min column YMC-Pack CN-AP, balanced pre 15% Century After loading the sample the concentration of propanol was increased from 15% to 25% and elution was performed with a linear gradient from 25% to 50% for 65 minutes, collecting eluate in polypropylene test tubes, 1.5 ml (2.5 minutes).

0,5 µl (1/3000 fraction) of the eluate in each tube was mixed with HSA, and was concentrated by ultrafiltration, receiving 0.25 ml IMDM test culture solution containing 0.05% S, for use in identifying TRO-active fractions using this test. In the heights 24-30 (in the range of concentrations of propanol 35,0 42,0 and%), which were United in the form of TRO-active fractions F (13,5 ml).

Fraction F obtained at the stage of the column YMC-Pack CN-AP was subjected to column chromatography on Cepcell Pak C1 300A (diameter 4.6 mm, height 150 mm, manufactured Shiseido, cat. C1 TYPE: SG 300A) with 0.1% F as a developing solvent a and 1-propanol containing 0.05% F, as developing solvent Century Part in (8.9 ml) fractions F obtained in column operations YMC-Pack CN-AP, was mixed with 0.3 ml of glycerol, concentrated by evaporation by centrifugation and then mixed with approximately 2.5 ml of 10% C. the resulting solution was applied then, when the flow rate 0.4 ml/min on the column, Capcell Pak C1 300A, balanced pre-20% C. After loading the column elution was performed first with 20% for 15 minutes and then a linear gradient from 20% to 40% for 50 minutes, collecting eluate in polypropylene test tubes and 1 ml (2.5 minutes) in each tube.

Part 1 ál (1/1000 fraction) of the eluate in each tube was mixed with the HSA and was concentrated by ultrafiltrate, receiving 0.25 ml IMDM test culture solution containing 0.05% HSA for use in identification TRO-active fractions of the test. As a result of high activity of TPO (relative activity preamture were United in the form of high-activity fraction.

Example 37. Measurement mol. mass TRO person, expressed in COS 1 cells is

An assumption was made that they say. weight full-TRO person (obtained from expressing plasmids pHTP1, clone F1) expressed in COS 1 cells, must be greater than the mol. mass, estimated on the basis of the size of the peptide chain, due to the addition of sugar chains. Therefore, as a first stage has been partially purified fraction TRO as follows from the culture supernatant containing the full-TRO person (obtained from expressing plasmids pHTP1, F1 clone) expressed in COS 1 cells. I.e. using developing solvent A (0.1% of triperoxonane acid (F) and exhibiting solvent (1-propanol containing 0.05% F) portion of 0.3 ml of the culture supernatant was applied on the column, YMC-Pack PROTEIN-RP (diameter and 0.46 cm, height of a layer 15 cm, UMC, cat. A-RR-33-46-25), balanced pre-25% C. 25% was passed through the column for 5 minutes at a flow rate of 0.4 ml/min and then performed fractionation using a linear density gradient from 25% to 50% within 50 minutes. TPO activity was detected in the eluates in the range of concentrations of propanol to 34.5% and 43.5%. These eluate Of activity was defined M-e test system after extraction of the protein from the gel SDS-PAGE electrophoresis, conducted with non conditions as described in example 1 or mol. weight was measured based on the reconstructed DPC111 markers by Western analysis, which will be described below in example 45. In the TPO activity was found in a wide range of average mol. masses of approximately 69000 up to 94000, indicating vazirani in mol. mass. It was also measured that the average mol. the mass of the TRO on the basis of the restored DRC markers after hydrolysis of the sugar chain-type N-glycosidic bonds using N-glycanase (Genzyme, cat. 1472-00) was 36000-40000, i.e., more than they say. weight of approximately 35,000, estimated from the size of the peptide chain, which is a strong indication of the presence also of a sugar chain type O-glycosidic bonds.

Similarly, it was found that the average mol. weight full-TRO person (obtained from expressing plasmids pHTP1, P1 clone), Expressivo in COS 1 cells, equal 63000-83000.

Example 38. Biological characteristics SRAW man

Influence TRO person on hematopoietic cells of humans and rats was investigated mainly using culture supernatant from COS 1 cells transfected pHTF1.

In the test by counting the colony is ka stimulated the formation of a significant number of colonies of megakaryocytes. For example, 11.5 colonies of megakaryocytes was formed 6000 CD34+DR+cells in the presence of 10% (vol./about.) the culture supernatant of COS 1 cells transfected with the pHT1-231. Test steps TRO person on precursor cells of human megakaryocytes received CD34+cells from peripheral human blood as follows. The fraction of cells derived from human peripheral blood using density gradient Ficoll-Paque, overburden from cells adherent to plastic cups. Unattached cells were then purified from the SBA (agglutinin soy)-positive cells penninga using AIS Microselecter SBA (Asahi Medical. The obtained cells were incubated at AIS Microselecter CD 34 and adsorbed cells, CD34+cells were collected after culturing CD34+cells for 10 days in the presence of culture supernatant from transfected COS 1 cells in a liquid medium, the observed selective cell proliferation Gp11b/111a+large size and found an increase in the ploidy in these Gp11b/111a+cells. These results provide a strong basis for assuming that the TRO person has a selective effect on precursor cells of human megakaryocytes and stimulates them about bogdanich CFU-MK from bone marrow cells of rats and of cells adherent to plastic, obtained at the previous stage Gp11b/111a+cells TRO human induced the formation of a significant number of colonies of megakaryocytes. For example, 34.5 colonies of megakaryocytes formed from 1000 Gp11b/111a+cells and 28.5 colonies megakaryocytes were formed 20000 not adherent to plastic cells in the presence of 20% (vol./about.) culture supernatant from transfected COS 1 cells. In addition, although the fraction is not attached to the plasticity of the cells contains a variety of hematopoietic precursor cells, other than CFU-MK in the presence of TPO man was formed only colony of megakaryocytes, suggesting that TRO person manifests its selective effect on precursor cells megakaryocytes origin. Under cultivation Gp11b/111a+cells derived from rat bone marrow, within 3-5 days in the presence of 20% (vol./about.) culture supernatant from transfected COS 1 cells in liquid culture, on the 5th day of cultivation observed an increase in the ploidy of the cells.

Example 39. Construction of vector for use in the expression of fused protein (hereinafter referred to as "GST-TPO (1-174)" glutathione-S-transferase (GST) and TRO cheloveki gene encoding TRO person and containing the codon preferences of E. coli. The nucleotide sequence of this DNA contains the amino-terminal methionine codon (ATG) at position -1 for use in initiation of translation in E. coli.

Synthetic oligonucleotides 1-12, are presented in table.IV, were prepared and each of the synthetic oligonucleotides 2-11 was fosforilirovanii T4 kinase (manufactured by Pharmacia) in a solution consisting of 1 mm ATP, 10 mm Tris-acetate, 10 mm MD-acetate and 50 mm K-acetate. To turn these synthetic oligonucleotides in 6 double-stranded DNA fragments were combined in combinations 1 and 2 3 and 4 5 and 6 7 and 8 9 and 10 11 and 12 and each combination was subjected to annealing in a solution consisting of 10 mm Tris-model HC1 (pH 7.5), 10 mm MgCl2and 50 mm NaCl. Then 3 double-stranded DNA fragment 1 and 2, 3 and 4 and 5 and 6 and 3 other double-stranded DNA fragment 7 and 8 9 and 10 11 and 12, respectively, and treated with T4-ligase (manufactured by Life Technology) and these 2 reaction solution was again treated with T4-ligase in the same way. The DNA fragment obtained by the ligation reaction was digested BamH1 (Boehringer-Mannheim) and subjected to electrophoresis in 2% agarose gel, removing the fragment about 390-400 p. N. in size, which is then cleaned using the cleaning set D Is Of the thus obtained clones clone, having an artificial gene that encodes a TRO person and contains the following preferred codons of E. coli was chosen as the analysis of the nucleotide sequence and was named pUC18(XB) (1-123). The DNA sequence coding circuit shown in the list of sequences (SEQ ID No 9) (see tab.V).

Single-stranded cDNA was synthesized from 1 µg obtained from normal human liver poly (A)+RNA using oligo dT-primer, and PCR was carried out using 0.1 volume of reaction solution for the synthesis of cDNA as template. PCR (incubation at 96oC for 2 minutes, repeat and, in General, 30 cycles each of which consisted of incubations at 96oC for 1 min, at 58oC for 1 minute and at 72oC for 1 minute, and the ending inkubirovanie with 72oC for 7 minutes) was performed with a volume of 100 μl using hTPO-C and hTPO-Z (EcoR1) as primers. The cDNA fragment TRO person, thus obtained, were digested BamHI and EcoR1 and subjected to electrophoresis in 2% agarose gel, removing the fragment size of about 600 p. N., which was then purified using the kit for purification of DNA Prep-A-Gene and was subcloned into pUC18 cleaved pre-EcoR1 and BamH1 (E. coli DH5 was used as ateneu sequence and he was named UC18(BE)(124-332). Oligonucleotide sequences of primers used in this R, were as follows:

. hTPO-C: 5'-GGA GGA GAC CAA GGC ACA GGA-3' (SEQ ID No 95) (position 329-349 clone hHTF1); and

. hTPO-Z(EcoR1): 5'-CCG GAA TTC TTA CCC TTC CTG AGA CAG ATT-3' (SEQ ID 96) (obtained by adding a sequence of recognition to the antisense primer corresponding provisions 1143-1163 clone pHTF1).

Clone pUC18(BE) (124-332) were digested ml and EcoR1 and subjected to electrophoresis in 2% agarose gel to extract the fragment C-terminal side of the cDNA TRO person approximately 600 p. N., which was then purified using the kit for purification of DNA Prep-A-Gene and was subcloned into pUC18(XB) (1-123), split pre-restrictable EcoR1 and BamHl (E. coli DH5 was used as the host). The clone thus obtained was named pUC18(XE)(1-332).

Because the presence of TPO activity in peptide fragment TRO person from the provisions of amino acid residues 1-163 was confirmed by the example, which were obtained by different C-terminal deletion constructs cDNA TRO person and expressed for measuring in vitro TPO activity was constructed expressing a vector containing this peptide fragment. In this case held the expression of posledovatelnosti-686 (amino acid residues 173 and 174) clone pHTF1, recognized by the restriction enzyme S1, two end codon was introduced using 2 synthetic oligonucleotides using site recognition of this restrictase. I.e., two synthetic oligonucleotide SSE1 and SSE2 annealed in a solution consisting of 10 mm Tris-model HC1 (pH 7.5), 10 mm MgCl2and 50 mm NaCl, and set for ligating DNA (Takara Shuzo) thus obtained double-stranded fragment was inserted in pUC18(HEH)(1-332), from which the C-terminal fragment of approximately 480 p. N. cDNA SRAW man was removed by splitting it Sacl and EcoR1, getting a clone U18(XS)(1-174) (E. coli DH5 was used as the host). Sequences of synthetic oligonucleotides used were as follows:

SSE1: CTAATGAG (SEQ ID No 97)

SSE2: AATTCTCATTAGAGCT (SEQ ID No 98);

< / BR>
. 5'-CTAATGAG-3' (SEQ ID No 99)

3'-TCGAGATTACTCTTAA-5' (SEQ ID No 100)

.

Clone pUC18 (XS) (1-174), obtained above, was digested Xbal and EcoR1 and subjected to electrophoresis in 2% agarose gel to extract the fragment size of about 600 p. N., encoding amino acid residues 1-174 TRO person. Then, this fragment was purified using the kit for purification of DNA Prep-A-Gene and was subcloned into pBluescript 11SK+(made Stratagene), pre-split Xbal and EcoR1 (E. coli DH5 was used as hose vlaka fragment of approximately 550 p. N., encoding amino acid residues 1-174 TRO person, and then this fragment was purified using the kit for purification of DNA Prep-A-Gene and was subcloned into the F536 (EP-A-136490), split pre-Xbal and Hind111 (E. coli DH5 was used as the host). The clone thus obtained was named FM536/h (1-174).

The following experiment was performed for the expression of GST-TPO(1-174) using the pGEX-2T (Pharmacia), which is expressing vector fused with glutathione - S-transferase (GST) protein. R (incubation at 96oC for 2 minutes, repeating as a whole 22 cycles, each of which consisted of incubations at 96oC for 1 minute, 41oC for 1 minute and at 72oC for 1 minute, and final incubation at 72oC for 7 minutes) was carried out using the F536/h(1-174) as a matrix and two PCR primers GEZ1 and GEX3. Encoding TRO human fragment thus obtained was digested Nl and EcoR1 and subjected to electrophoresis in 2% agarose gel, extracting a fragment of approximately 550 p. N., this fragment was then purified using the kit for purification of DNA Prep-A-Gene and cloned in the G-2T, split in advance EcoR1 and Smal (E. coli DH5 was used as the host) and then clone named pGEX-2T/hT(1-174) was from GST-TRO (1-174). Oligonucleotide sequences used primers in the PCR were as follows:

GEX1: 5'-GCC ATC GGC TCC GCC AGC TTG TGA C-3'-(SEQ ID No 101) (obtained by adding a sequence of recognition Nael to the sequence of the provisions 21-39 in SEQ ID 10) and

. G3: 5'-GCC GAA TTC TCA TTA GAG CTC GTT CAG TGT-3' (SEC ID 102) (antisense primer corresponding to the sequence positions 523-549 in SEQ ID 10).

This expressing plasmid contains a DNA sequence encoding a GST protein, followed by peptide recognition by thrombin and SRAW man (amino acid residues 1-174). The DNA sequence encoding the peptide recognition by thrombin and SRAW man (amino acid residues 1-174), shown in the list of sequences (SEQ ID 10).

Example 40. Expression of GST-TPO (1-174) in E. coli

The transformant obtained in example 39, were cultured overnight at 37oWith on the shaker using 60 ml of LB medium containing 50 μg/ml ampicillin, and 25 ml of the obtained culture broth was added to 1000 ml of L medium containing 50 μg/ml of ampicillin, and cultured at 37oOn the rocking chair, while CD AND when600he reached to 0.7-0.8. After that 1PTG was added to the culture broth to a final concentration of 0.1 mm and swing culture was continued for another 3 cha is odtwarzanie its biological activity

5.9 g of frozen cells of the recombinant strain producing GS-TRO (1-174) derived from clone pGEX-2T/hT (1-174), the coding nucleotide sequence SRAW man, suspended in 10 ml of water and destroyed the cage mill high pressure. After centrifugation the resulting suspension GST-TRO (1-174) was recovered in the besieged faction, most of the impurity proteins, cellular components, etc., were removed. Then extracted thus precipitated fraction containing GST-TPO (1-174), suspended in 5 ml of water, to which was then added with stirring, 6 ml of 1M Tris buffer (pH 8.5), 120 ml of 10 M urea and 16 ml of water. After 5 minutes stirring to dissolve the contents of the resulting solution was evenly divided into 4 parts for carrying out the following steps (1) to(4).

(1) One of the samples was diluted 10 times in 20 mm Tris buffer (pH 8.5). Then add the restored glutathione and glutathione oxidized type to the appropriate final concentration of 5 mm and 0.5 mm, followed by incubation overnight at 4oC. the resulting mixture was centrifuged to extract GST-TPO (1-174) supernatant liquid, which was then diluted 2 times with 20 mm sodium citrate buffer (pH 5.5) and then brought the pH to 5.5 with acetic who novachannel pre 20 mm buffer of sodium citrate (pH 5.5). After washing the column 20 mm sodium citrate pH 5.5, the elution of GST - TRO (1-174) was performed in 20 mm buffer containing sodium citrate, pH 5.5, containing 500 mm sodium chloride. Part 129 ml of the eluate was mixed with 2.6 ml of 1 M Tris buffer, pH 8.5 and the resulting mixture with a pH of 8.1 was applied to a column of Glutathione Sepharose 4B (Pharmacia Biotech, cat. 17-0756-01) for adsorption of GST-TPO (1-174). After washing the column PBS was performed elution of GST-TPO (1-174) using Tris-buffer, pH 8.5, containing 10 mm glutathione restored type. The obtained eluate was mixed with 37 NlH units of thrombin, was allowed to stand for 4 hours at room temperature, was diluted 10 times in PBS and then were applied to a column of Glutathione Sepharose 4B for adsorption derived GST and extract SRW (1-174) in readsorbing faction. Retrieved readsorbing fraction was diluted 3 times containing 20 mm sodium citrate buffer with a pH of 5.5 and was applied to a cation exchange column, SP Sepharose Fast Flow equilibrated previously with the same buffer, and the target compound was suirable linear density gradient 0-500 mm sodium chloride, dissolved in the same buffer.

(2) All operations stage (1) was performed in the presence of 0.1% Polysorbate 80.

(3) One of the samples was diluted 10-fold to 20 mm Trio-buffer with a pH of 8.5. To it was added g of 0.5 mm, followed by standing overnight at 4oC. the resulting mixture was centrifuged to extract GST-TPO (1-174) supernatant liquid, which was then diluted 2 times containing 20 mm sodium citrate buffer with pH 5.5 and then brought the pH to 5.5 with acetic acid. GST-TPO (1-174) in this solution was applied to a cation-exchange resin SP Sepharose Fast Flow, pre-uravnoveshennoy containing 20 mm sodium citrate buffer with a pH of 5.5. After washing the resin containing 20 mm sodium citrate buffer with a pH of 5.5 was performed elution of GST-TPO (1-174) containing 20 mm sodium citrate buffer with pH 5.5, containing 500 mm sodium chloride. The eluate was brought to pH 8, 1 M Tris-buffer pH 8.5, was mixed with 320 NlH units of thrombin to remove the polypeptide sequences of GST by cleavage at the peptide linker recognition of thrombin and allowed to stand for 4 hours at room temperature, was diluted 5-fold containing 20 mm sodium citrate buffer with pH 5.5 and then was applied to a cation exchange resin SP Sepharose Fast Flow equilibrated in advance with the same buffer, and the target compound was suirable then a linear density gradient 0-500 mm sodium chloride, dissolved in the same buffer.

(4) All operations stage (3) was performed in the presence of 0.1% Polysorbate 80.

Each of the fractions, the constant column of SP Sepharose Fast Flow, thoroughly were dialyzed against IMDM culture medium and then evaluated in the test system rat CFU-MK. When analyzing the fractions by electrophoresis SDS-PAGE in the presence of the reducing agent detected a band corresponding to the mol. weight of 19 KD (purity 1-20%) as one of the major protein bands of this fraction. In the analysis of N-terminal sequence of this band in SDS-PAGE and transfer to PVDF membrane according to the method described in example 1, it was confirmed that this strip contains the sequence TRO expressed in the form derived from pGEX-2T/hT(1-174) fused protein. Deduced (derived) amino acid sequence resulting TRO was [Gly-1] TPO due to the sequence of peptide linker recognition of thrombin and known activity of thrombin on the linker.

Example 42. Construction of vector for expression in E. Li TRO person (amino acid residues 1-163) having a substitution in position 1 (Ser-->Ala) and position 3 (Ala-->Val)

Clone pUC18(XE) (1-332) obtained in Example 39, Xbal digested and EcoR1 and subjected to electrophoresis in 2% agarose gel, releasing a fragment size of about 1000 p. N., encoding amino acid residues 1-332 TRO person, and this piece is th Stratagene), which was split in advance by restrictase Xbal and EcoR1 (E. coli DH5 was used as the host). The clone thus obtained was named pBL(XE)(1-332). Then the area of this clone pBL (XE)(1-332) from its sequence BamH1 recognition to the position of 163 amino acids (position 366-489) was replaced by the dominant E. coli codons. Were obtained synthetic oligonucleotides 13-20, shown below, and each pair of synthetic oligonucleotides 13 and 14, 15 and 16 and 17 and 18 were fosforilirovanii in the same test tube containing a solution consisting of 0.1 mm ATP, 10 mm Tris-acetate, 10 mm MD-acetate and 50 mm K-acetate. After further adding 1/10 volume of a solution consisting of 100 mm Tris/Hcl (pH 7.5), 100 mm MgCl2and 500 mm NaCl, and the resulting solution was heated in a boiling bath for 3 minutes and then allowed to cool to room temperature, obtaining a double-stranded DNA fragment. Then thus obtained three pairs of double-stranded fragments, consisting of 13 and 14, 15 and 16 and 17 and 18, ligated with a set for ligating DNA (Takara Shuzo) and PCR was performed using the very product as template and synthetic oligonucleotides 19 and 20 as primers. The resulting PCR product was digested BamH1 and Hind111 and subjected to electrophoresis in 2% agarose gel, which was subcloned into the pBL(XE)(1-332), split pre Wamn and Hind111 (E. coli DN used as a master). The clone having the nucleotide sequences shown in table. VI, was selected from the obtained clones analysis of the nucleotide sequence and named pBL (XH)(1-163).

With the aim of increasing the number of expressed TRO person (1-163) and prevent hydrolysis of the N-Terminus of the protease was constructed expressing vector for use in the expression of the mutated type SRW person (1-163) (hereinafter called "h 6T(1-163)") having a substitution at position 1 (Ser to Ala) and position 3 (Ala to Val) TRO person (1-163) ([l1, Val3] TRO (1-163)) and, at the same time, encoding Lys at the -1 position and Met at the -2 position ([C-2, Lys-1Ala1, Val3] TPO (1-163)). Received four synthetic oligonucleotide shown below, and synthetic oligonucleotides 2-9 and 3-3 were fosforilirovanii T4 kinase (manufactured by Pharmacia) in a solution consisting of 1 mm ATP, 10 mm Tris-acetate, 10 mm MD-acetate and 50 mm K-acetate. In order to make these synthetic oligonucleotides to double-stranded DNA fragments, each pair of single-stranded DNA fragments 1-9 and 2-9 and 3-3 and 4-3 were annealed in a solution consisting of 10 mm consisting of 1-9 and 2-9 and 3-3 and 4-3, were processed using the set for ligating DNA (Takagi Shuzo). The DNA fragment obtained in the reaction of ligation, was subcloned into the pBL(XH)(1-163), split pre-Xba1 and Nru1 and the clone correctly replaced by the following synthetic oligonucleotide shown in table. VII, was selected by analysis of the nucleotide sequence (E. coli DN used as a master) and named BL(HN)h6(1-163).

Clone L (XH)(1-163) Xba1 digested and Hind111, extracting a fragment of approximately 500 p. N., encoding amino acid residues 1-163 TRO human mutated type, and this fragment was then purified using the kit for purification of DNA Prep-A-Gene and cloned in F536 (EP-A-136490), split pre-Xba1 and Hind111 (E. coli JM109, which was previously transformed pMW1 (ATSS 39933), was used as the host). The clone thus obtained was named F536/h6 (1-163), and an E. coli strain that expresses the vector, used as transformant for use in the expression of TPO human mutated type, namely h6T(1-163). This expressing plasmid contains the DNA sequence shown in the list of sequences (SEQ ID 11).

Example 43. Expression h6 (1-163) in E. Li

Expression of expr is on s. The transformant obtained in Example 42, were cultured overnight at 30oWith on the shaker using 60 ml of LB medium containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline, and part 25 µl of the obtained culture broth was added to 1000 ml of L medium containing 50 μg/ml of ampicillin, and cultured at 37oWith the shaker up until OD at A600he reached 1,0-1,2. After approximately 330 ml of L medium heated to 65oC, was added to the culture broth, bringing the final temperature of the environment to the 42oWith, and swing culture was continued for 3 hours at 42oWith to induce the expression of h6(1-163).

Example 44. Cleaning h6(1-163), expressed in E. Li, and confirmation of its biological activity

A portion of 3.6 g of frozen cells h6T(1-163) producing recombinant strain suspended in 10 ml of water and was destroyed with the help of the cage mill high pressure. Centrifugation of this suspension has been besieged faction, and most of the impurity proteins, cellular components, etc., were removed. Retrieved precipitated fraction containing h6(1-163), suspended in 7 ml of water and to the suspension under stirring was added 3 ml of Tris-buffer (pH 8.5), followed EXT is the (final concentration 2%). After 5-20 minutes of stirring at room temperature for the dissolution of the components of the resulting solution was diluted 10 times in 20 mm Tris-buffer pH 8.5 and subjected to repeated protein laying chain by incubating overnight at 4oC. In this case, the used glutathione and copper sulfate as an additive, in addition to air oxidation. By centrifugation 6T(1-163) extracted from supernatant fluid. When analyzing each of the extracted fractions using SDS-PAGE in the presence of the reducing agent detected a band corresponding to the mol. the mass of 18 KD (purity 30-40%) as the main protein bands in each fraction. In the analysis, N-terminal sequence using SDS-PAGE and transfer to PVDF membrane according to the method described in Example 1, it was confirmed that this band contains expressed sequence TRO in the form received from F536/h6(1-163) TRO mutated type. With careful dialysis each fraction against 1MDM culture medium and evaluation using the test system rat FU-MK TPO activity was detected in all fractions, and it was dose dependent.

Example 45. Obtaining antibodies against TPO peptide and detection TRO using elektropotreblenie protein TRO immunological method. In this regard, three of the district, which, apparently, are relatively applicable as antigens (see below), were selected from proven amino acid sequence TRO and peptide type quadrupole-chain peptide with multiple antigens (MAP) was synthesized according to the method Tam(Proc.Natl Acad.Sci.USA, 85, 5409-5413, 1988) using each of the selected areas and then each of 2 rabbits were immunized 8 times with 100 μg of each of the synthesized thus peptides to obtain the appropriate samples antisera.

Amino acid sequence contained in the synthesized peptide antigens

(a) Rat TPO (9-28) (Peptide region R T1)

< / BR>
( at position 24 is the source S in amino acid residues that are defined on the basis of the gene)

(b) Rat TPO (46-66) (Peptide district RT2)

FSLGWKTQTEQSKAQDILGA (SEQ 1D 117)

(C) Rat TPO (163-180) (Peptide district RT4)

< / BR>
( provisions 178-180 source is a TSG in amino acid residues that are defined on the basis of this gene)

Then these samples antisera, anti-RT1 peptide and anti-RT2 peptide was subjected to first column chromatography on Protein A (PROSEP-A, Bioprocessinq Ltd. , cat. 8427) to obtain the receiving them active Biotin (NHS-LC - Biotin 11; izgotovliaemye P1ER CE, cat. 21336). Recombinant TPO-containing sample was subjected to SDS - PAGE and then electroblotting on PVDF or nitrocellulose membrane as described in Example 1. Western analysis was carried out in the usual way using these biotinylating antibody as the first antibody.

I.e. the membrane after blotting was washed with a solution consisting of 20 mm Tris-HC1 and 0.5 N1 (pH 7.5) (S) for 5 minutes and twice with 0.1% tween 20-containing S (S) for 5 minutes each time and then was treated with a blocking agent (Block ACE; manufactured by Dainippon Pharmaceutical, cat. IR-B25) for 60 minutes. Then the membrane was treated for 60 minutes with a solution S containing 10 μg/ml biotinylated antibodies against peptide TPO, 0,05% S and 10% BlockAce and then washed twice S each time for 5 minutes. After that, the membrane was treated for 30 minutes with a solution obtained by diluting the labeled alkaline phosphatase avidin (Leinco Technoloqies, cat. A108), 5000 times with a solution S containing 10% BlockAce, and washed twice for 5 minutes with TTBS and S within 5 minutes. After this was completed the development of painting using a substrate of alkaline phosphatase (manufactured by Bio-Rad, cat. 170-6432). Described Western analysis was carried out at room temperanceville in COS 1 cells, but also recombinant human proteins TRO expressed in COS 1 cells and E. coli cells (e.g., obtained from plasmid rntr or pHTF1 TRO person, expressed in OS 1 cells, and the product obtained by the cleavage of N-glycosidic bonds, GS-TRO (1-174), expressed in E. coli and cleaved by thrombin product and h6(1-163), which is TRO mutated type, expressed in E. coli, which were described in the Examples). In addition, these results gave us the opportunity to analyze these different types of recombinant TPO person.

Besides the above, was confirmed by the possibility of obtaining antibodies against peptide TPO person. Antibodies obtained by these methods can be applied not only for Western analysis, but also for cleaning TRO using columns with antibodies and any conventional immunological methods using antibodies.

6 regions in amino acid sequences TRO person shown in SEQ 1D 7, which, as expected, have a relatively suitable antigenicity, were selected (see Table 4). Were synthesized peptides type tetramer peptide with multiple antigens (MAP) corresponding to these areas. Each peptide were immunized 2 is epted, in which a cysteine residue has been associated with the end of each peptide region, shown in Table 4. This peptide is then used as the test antigen to determine the titer of antibodies using enzyme immunoassay. As a result, the prepared serum was shown to increase antibody titer, so these sera were used as antisera.

Antibodies were obtained by immunization of each antigenic peptide of two rabbits for the production of antisera directed against this peptide. Received two antibodies against peptide NT to differentiate called antibody against the peptide NT-1 and the antibody against the peptide NT-2, as obtained in two separate rabbits.

An example of the purification of antibodies against peptide NT 1 will be shown below.

First, 30 mg of Monomeric peptide NT connected to the cysteine residue, was associated with 12 ml Sulfo-Link coupling gel (Pierce, cat. 44895). Briefly, a solution of a peptide containing the antigen, bound for 15 minutes with gel, balanced binding buffer (50 mm Tris, 5 mm EDTA-Na, pH 8.5) in the amount of 6 volumes to the volume of the gel. After incubation for 30 minutes, the gel was washed svyzivautsy buffer in an amount of 3 volumes on byvania unreacted radicals within 15 minutes. After incubation for 30 minutes, the gel was washed in binding buffer (8 volumes to 1 volume of gel). All binding assays were carried out at room temperature. Thus, the peptide was covalently linked to gel with the binding efficiency of 28.3%, and was prepared column with antigen containing 0.8 mg of peptide per 1 ml of gel.

76,7 ml (protein 3620 mg) antisera containing an antibody against the peptide NT-1, which was obtained from one of the rabbits in the total number 78,4 ml, was applied on the column with the antigen pre-equilibrated with 50 mm phosphate buffer (pH 8) containing 150 mm N1 and 0.05% sodium azide, and then washed with the same buffer, receiving 105,9 ml passing through the column fractions (protein 3680 mg). Then suirable adsorbed fraction 0.1 M citrate buffer (pH 3.0) and immediately neutralized to 21.1 ml of 0.1 M carbonate buffer (pH 9,9), followed by concentration by ultrafiltration with the use of membrane Amicon MIND 30), receiving the purified antibody against the peptide NT-1 in a solution of 50 mm phosphate buffer (pH 8) containing 150 mm Nl and 0.05% NaN3.

In this way received the following antibodies: antibody against peptide NT-2 (60,0 mg); antibody against the peptide NT2-1 (18

3 mg of affinity-purified antibodies against peptide NT-1, against a peptide NT-2, against peptide NT2-1 or anti-peptide NT2-2 was biotinilated binding with activated Biotin (Pierce, NHS-LC-Biotin 11, cat. 21336).

It was found that all of the above purified antibodies recognize and detect SRAW man in the analysis of Western blot testing (on a nitrocellulose filter or PVDF) after SDS-PAGE of recombinant standard SRW man, partially purified from the culture supernatant of Cho cells into which has been introduced a gene encoding the amino acid sequence shown in Table 4, and in which the gene expressively.

Table 4

Amino acid sequence TRO person included in the synthetic peptide antigen type tetramer MAP

Peptide district NT TRO person (number of amino acids 8-28): DLRVLSKLLRDSHVLHSRLSQ (SEQ 1D 119)

Peptide district NT2 TRO person (number of amino acids 47-62): SLGEWKTQMEETKAQD (SEQ 1D 120)

Peptide district NT3 TRO, person (number of amino acids 108-126): LGTQLPPQGRTTAHKDPNA (SEQ 1D 121)

Peptide district NT TRO person (number of amino acids 172-190): NELPNRTSGLLETNFTASA (SEQ 1D 122)

Peptide district NT TRO person (number of amino acids 262-284): SLPPNLQPGYSPSPTHPPTGQYT (SEQ 1D 123)

Peptide Paradise is itelli against peptides TRO

Because the antibody against the peptide SRAW rats, obtained in Example 45, was able to identify molecules rat and human the solid fraction of immunoglobulin (1gG) antibodies against peptide RT1 and antibodies against peptide RT2 were connected with chemically activated gel media for making columns with antibodies against peptides TRO as follows. Each of the two antibodies used as such materials were obtained from sera of two rabbits separately. I.e., antibodies against peptide RT1 obtained from two rabbits were respectively named antibody against peptide R1-1 and an antibody against the peptide R1-2, and antibodies against peptide R2 obtained from the other two rabbits were respectively named antibody against peptide RT2-1 and an antibody against the peptide RT2-2. Because these antibodies were used separately to prepare columns with antibodies, received only 5 columns, namely, two columns with antibodies against peptide RT1 (hereinafter called the column with anti-RT1-1 antibodies and a column with anti-RT1-2 antibodies), two columns with antibodies against peptide RT2 (hereinafter called the column with anti-R2-1 antibodies and a column with anti-RT2-2 antibodies) and one column (the column with a mixture of antibodies anti-RT1--2+2-1)/P> Each antibody was dissolved in a solution of 50 mm of sodium phosphate and 0.15 M N1 (pH 8.0) to a final concentration of 5 mg/ml or 2.5 mg/ml each of evenly mixed anti-RT1-2 antibodies and anti-R2-1 antibodies in the case of the column with a mixture of anti-RT1-1+2 antibodies) and 2,31 ml of the resulting solution was mixed with and 1.54 ml in volume of the swollen formyl-activated gel (Formyl-Cellulofine, Chisso) and subjected to 2-hour binding assays at 4oC. Then was added 1.1 ml of a solution of 10 mg/ml reducing agent (trimethylaminuria (TMAU); machines are manufactured by Seikagaku Kogyo, cat. 680246), followed by 6 hours of additional binding assays and subsequent centrifugation to extract the gel. 10 ml of purified water was added to this portion of the gel, again centrifuged and unreacted molecules of antibody was removed by repetition of this stage 4 times. Then, the resulting gel was mixed with 4.6 ml of a blocking solution (0.2 M sodium phosphate and 1 M ethanolamine, pH 7.0) and 1.1 ml of a solution of a reducing agent and treated at 4oWith at least 2 hours to block unreacted active groups of the gel. After that, the gel was washed with purified water and DPBS using centrifuges, Packed in a tube to a small column, washed with 3 M rastah with antibodies against peptides TRO column with anti-RT1-1 antibodies columns with anti-RT1-2 antibodies, speakers with anti-RT2-1 antibodies, speakers with anti-R2-2 antibodies and columns with a mixture of anti R1-2+2-1 antibodies, the binding efficiency of the corresponding fractions lgG reached 97,4%, 95,4%, 98,4%, 98,3% and 99.4%. In addition, the number of fractions lgG related to the volume of the gel, were 5.6 mg/ml of gel, and 5.8 mg/ml gel, 5,7 mg/ml gel, 5,7 mg/ml gel and 2.9 mg/ml of gel, respectively. Therefore, since it was confirmed that the efficiency of binding and the number of binding has not changed significantly depending on the origin of antibodies and differences in the antigens, the experiments shown in the following Example 47, was performed using these speakers with antibodies.

Example 47. Cleaning TRO from the culture supernatant obtained by transfection of OS 1 cells expressing vector rntr, using speakers with anti-TPO antibodies and subsequent column chromatography with reversed phase and the confirmation of its biological activity

For evaluation of the following purified samples were used M-e the test system as a test in vitro. Partially purified samples TRO from the culture supernatant obtained in Example 35 using 1 COS cells transfected expressing vector rntr (fraction other than onoria, pH of 5.8, column Macho-Rger Methyl H1C and fractions F1 and F3 column of SP Sepharose Fast Flow) were combined to obtain a fraction subpool TRO (5453,79 ml; protein concentration 0,490 mg/ml; total protein 2676 mg; relative activity 12100 and General activity 32380000) and concentrated using ultrafiltration devices (Omega Ultraset, nominal clipping mol. weight 8000, manufactured Filtron), the solvent is concentrated fraction was replaced with DPBS and then received a small sample volume processed by ultrafiltration device with a UM-10 membrane (Amicon), receiving at the end of the sample 120,2 ml DPBS containing 0.05% of sodium azide. Then all 5 of gels with antibodies against peptides of the solid obtained in Example 46, was mixed and prepared a single column with antibodies (diameter 1.6 cm and a height of 4.8 cm, called column a mixture of anti-R1+2 antibodies). Fraction TRO subpool were applied to the column at a rate of flow 0,033 ml/min After the download is complete, the column elution was performed DPBS to collect eluates up until the UV absorption was not low enough. Thus collected eluate concentrated by ultrafiltration with a UM-10 membrane (Amicon), receiving passing through the column fraction F1 (86,62 ml; protein concentration of 14.3 mg/ml; total bell; the protein concentration of 0.12 mg/ml; total protein of 11.1 mg; relative activity 257000 and General activity 2860000) was suirable acidic eluting solution (0.1 M glycine-model HC1, pH of 2.5). While passing through the column fraction F1 still contained TRO activity adsorbed by the column with TPO antibodies were purified further, because he had found an increase in the relative activity of about 20 times, i.e., with 0.1% TFA as a developing solvent a and 1-propanol containing 0.05% F as a developing solvent In column chromatography was performed on a Capcell Pak CL 300A (Shiseido, cat. C1 TOURS: SG 300A diameter of 4.6 mm and a height of 150 mm, which is connected with the pre-column with a diameter of 4.6 mm and a height of 35 mm) by mixing fractions F2, obtained from the column with antibodies, with 1/10 volume of manifesting solvent and applying the mixture at a flow rate of 0.4 ml/min on the column, Capcell Pak C1 300A, balanced in advance 20% of the Century. After the download is complete, the column elution was performed within 5 minutes, 20% In and then for 50 minutes linear density gradient from 20% to 40% In and eluate collected in polypropylene tubes in portions of 1 ml (2.5 minutes).

Portions of 2 ml (1/500 fraction) of the eluate in each tube was mixed with HSA and was concentrated by ultrafiltration, oluranti this way. As a result of high TPO activity was found in samples of test tubes with numbers 20-23 (in the range of concentration of propanol and 28,5 32,5%). In addition, these samples were subjected to Western analysis according to the method described in Example 45, and was confirmed by the presence of TPO, which had an average mol. a lot 60000-70000 when measured under reducing conditions on the basis of DRC markers, mol. masses, and the presence of other molecules that have the appropriate mol. mass 32000-43000 and 20000 to 30,000.

Example 48 Confirmation of biological activity TRO from the culture supernatant obtained by transfection of COS 1 cells expressing vector rntr, and cleaned up the stage speakers pcl Pak C1 300A

TRO-active fraction purified in Example 36, namely, test tubes with numbers 20-23 (in the range of concentration of propanol and 28,5 32,5%) column, Capcell Pak C1 300A, combined, mixed with of 0.21 ml of glycerin and then was concentrated by evaporation under centrifugation. The concentrate was mixed with of 0.21 ml of a 6 M solution of guanidine hydrochloride, diluted to a volume of 1 ml DS, the buffer was replaced DS containing 0.01 % of the HSA using column Sephadex G-25 (NAP-10, Pharmacia Biotech, cat. 17-0854-01) and then mixed with 1.1 ml of a solution DS containing 0,01% S, getting in the TRO-aktivni was administered subcutaneously 1R to male mice (8 weeks of age), each group consisted of 4 animals daily for 5 days at a dose of 100 µl (with a total activity of 110,000 measured test system with M-e). As a control were injected 100 μl DS containing 0,01% S, subcutaneously. Just before the introduction and the next day after the last injection had collected the blood from the fundus of the eye to measure the number of platelets using microcamera (F800, Toa 1yo Denshi). In the treated TRO group in the number of platelets increased from an average of approximately 1.42 times after injection compared with the value before the introduction with a significant difference between values (p<0,05, Student t-test). On the basis of these results, it was confirmed that the above-mentioned SRAW man, produced by mammalian cells, are able to increase the number of platelets in vivo.

Example 49. Confirmation of biological activity of crude fractions of the solid obtained from 33 l of the culture supernatant obtained by transfection of COS 1 cells expressing vector rntr, and partially purified by cation exchange column

1) M-e test system was used as an in vitro test to evaluate the following purified samples. In the same way as described in Example 36, in General, 33 l passionately through the filter of 0.22 μm to obtain a supernatant. It was concentrated approximately 10-fold using ultrafiltration device (PLGC pellicon Cassette, nominal clipping mol. weight of 10,000, manufactured Millipore) and were mixed with 1 mm final concentration protease inhibitor p-APMSF, receiving the sample 2018 ml by volume (concentration of protein 3,22 mg/ml; total protein 6502 mg; relative activity 66000 and General activity 429100000). Then the volume was concentrated again using ultrafiltration devices (Omega Ultraset, clipping mol. mass 30000; Filtron), resulting in the fraction (mol. weight of 30,000 or more (by volume 1190 ml; protein concentration of 2.54 mg/ml; total protein 3020 mg; relative activity 82500 and General activity 249000000) and fraction (mol. weight of 30000 or less (volume 2947 ml; protein concentration 0,471 mg/ml; total protein 1402 mg; relative activity 4500 and General activity 6310000). The presence of TPO activity in fractions (mol. weight of 30000 or less indicates the possibility that this culture supernatant contains TRO, mol. weight decreased while its expression in animal cells or secretion from animal cells. On the other hand, the fraction of 30,000 or more after replacing the buffer is 20 mm Na-citrate buffer (6,1) was applied at a flow rate of 5 ml/min in 20 mm sodium citrate buffer (pH 6,1). After loading the sample the column was washed and was suirable 20 mm nitrate buffer (pH 6,1). Then, using 20 mm sodium citrate buffer (pH 6,1) as developing solvent and solution consisting of a developing solvent a and 1 M N1, as developing solvent elution was performed at a flow rate of 3 ml/min linear gradient from 0% to 50% In over 215 minutes and then from 50% to 100% within 20 minutes. Eluate collected 30 ml (10 min) in polypropylene tubes. When checking portions of each of these eluates in M-e-test the system to determine the distribution of activity elution of TPO activity was detected in a wide range. Therefore, fractions, erwerbende concentration N1 50 mm or below, including passing through the column fractions were pooled as fraction F1, and the main TRO faction, erwerbende 50-1000 mm N1, were United in the form of fraction F2. The volume fraction F1 was 1951 ml (protein concentration of 2.05 mg/ml; total protein 3994 mg; relative activity 13500 and General activity 53900000). The volume fraction F2 was 649,8 ml (protein concentration of 1.11 mg/ml; total protein 721 mg; relative activity 268000 and General activity 193000000).

2) Confirmation of biological activity of fraction F2 SP Sepharose Fast Flow

Example 50. Construction of recombinant vector, DEF202 - gh TRO, for use in the expression of chromosomal DNA TRO person in Cho cells

Vector pDEF 202 were digested with restrictase Kpn1 and S 1 and then subjected eleanya SV40, and expressing vector DF 202-gh TRO was obtained by legirovaniem using T4 DNA ligase (Takara Shuzo) thus obtained fragment with the vector DNA, which was obtained by removing containing the SV40 polyadenylation signal region from plasmid FG by processing restrictase CRP and S1. This plasmid contains the origin (the origin) replication of SV40, 1 - promoter of the elongation factor of the person, the area of early SV40 polyadenylation transcription of the gene SRW human, mouse DHFR of minigun, start replication pUC 18 and gene-lactamase (AMRr). Chromosomal DNA TRO person connected with the site in the direction 5'-3' 1 - promoter of the elongation factor of the person.

Example 51. The expression of chromosomal DNA TRO person in Cho cells

Cho cells (strain dhfr-; Urlaub and Chasin, Proc. Natl. Acad.Sci.USA, vol. 77, p. 4216, 1980) were grown by culturing them in a basic medium (-MEM(-), added thymidine and gipoksantin) containing 10% fetal bovine serum, using a Cup with a diameter of 6 cm (manufactured by Falcon), and the obtained cells were transfusional calcium-phosphate method (CellPhect, Pharmacia).

I.e. 10 μg of plasmid pDEF202-gh TRO obtained in Example 50, was mixed with 120 μl of buffer a and 120 μl of N2Oh and the resulting mixture incubate at room temperature for 30 minutes. Thus obtained DNA solution was placed in a Cup and were cultured for 6 hours in CO2-incubator. After removing the medium and washing the obtained Cup twice-MEM(-) was added MEME ( -) containing 10% dimethyl sulfoxide, to the Cup and processed for 20 minutes at room temperature. Then he added a non-selective medium (-MEM(-) with the addition of gipoksantina and thymidine) containing 10% cialisbuynow FCS, and cultured for 2 days and then were selected with the use of selective medium (-MEM(-), not containing gipoksantina and thymidine) containing 10% cialisbuynow FCS. The selection was performed by treating the cells with trypsin, the distribution of the treated cells in one Cup (diameter 6 cm) 5 cups with a diameter of 10 cm or 20 cups of type 24-well plates and the continued cultivation of these cells to change selective medium every 2 days. The presence of TPO activity was confirmed in the culture supernatant in these cups or holes, in which there was a proliferation of cells in the measurement of SRAW activity of a person using BA/F3 test.

In this case, the transfection of Cho cells can also be cotransfected Cho cells with plasmids FG and pMG1.

Example 52. Constr) (hereinafter called "h(1-163)", in which the remainder of the Lus and the remainder Met were respectively added to the positions -1 and -2, and the confirmation of its expression

For expression of the protein, amino acid residues whose positions 1 and 3 are the same as in the protein TRO person (amino acid residues 1-163), was constructed vector F536/h(1-163), also known as [Met-2, Lys-1] TRO (1-163), for use in expression h (1-163) in E. coli using the same method described in Example 42, using the following synthetic oligonucleotides 1-13, 2-13, 3-3 and 4-3, are presented in table.VIII.

This expressing plasmid contains the DNA sequence shown in the list of sequences (SEQ 1D 12). After this expression h ( 1-163) induce the same way as described in Example 43.

When processing the thus obtained downregulation of protein SDS-RADA, transfer to PVDF membrane and analysis of N-terminal amino acid sequence was confirmed that this protein contains the amino acid sequence h(1-163), which was to be expressed.

Example 53. Putting the chain TRO person, h6(1-163) obtained from SRW person, expressed in E. coli, using guanidine hydrochloride and glutathione and cleaning and p is anantnag strain, obtained in Example 43, suspended in 3 ml of water and was destroyed with the help of the cage mill high pressure. Centrifugation of this suspension of precipitated fraction was removed and most of the impurity proteins, cellular components, etc., were removed. Thus obtained precipitated fraction containing h6T(1-163), suspended in 4 ml final volume of water, 1 ml of 1 M Tris buffer (pH 8.5) was added to this suspension with stirring, followed by adding 20 ml of 8 M handinhand, followed by stirring for 5 minutes at room temperature to dissolve the contents. The resulting solution was diluted 10 times in 20 mm Tris buffer (pH 8.5), 5 mm glutathione recovered type and 0.5 mm oxidized glutathione was dissolved in diluted solution with stirring, and then the thus prepared solution was incubated overnight at 4oC. By centrifugation h6(1-163) was extracted in supernatant fluid. Portion 160 ml obtained supernatant liquid was concentrated using AN ultrafiltration membrane (Amicon) and the buffer was exchanged for DPBS, gaining 3.4 ml end volume fraction (protein concentration of 2.18 mg/ml). With careful dialysis this faction against 1 of MDM cultural the th activity 58000).

Obtained TRO-active fraction was subjected to testing in vivo. This fraction was injected subcutaneously 1R to male mice (7 weeks of age), and each group consisted of 4 animals daily for 5 consecutive days at a dose of 170 μl (having a total activity 22000, as measured in the test system rat CFU-MK) 1 pet. As a control 170 μl DPBS) was subcutaneously injected to each of the 6 animals in the control group in the same way. Just before the introduction and the next day after the last injection, blood was collected from the fundus of the eye to measure the number of platelets using microcamera (F800, machines are manufactured by Toa Iyo Denshi). In the group with the introduction of the TRO platelet counts increased by an average of approximately 2.15 times the next day after injection compared with the value before administration, and this effect remained unchanged even after 3 days after injection, showing 2.13 times higher value. Platelet count the next day and on the 3rd day after injection were 1.73 and of 1.80 times higher in the group treated with the solid than in the control group, with significant difference between them (p<0,001, Student t-test). These results showed that SRAW man, producere the example 54. Putting the chain TRO person, h6(1-163), expressed in E. coli, using N-lauroylsarcosinate sodium and copper sulfate and purification and confirmation of its biological activity.

Portions of 0.6 g of frozen cells producing h6(1-163) recombinant strain obtained in Example 43, suspended in 3 ml of water, was destroyed by the cage mill high pressure and then centrifuged to extract the precipitated fraction. After the suspension of settled solids in 3.1 ml of water, to 0.19 ml of 1 M Tris buffer (pH 9,2), 11,25 ál of 1 M DTT, 38 μl of 0.5 M EDTA and 0.38 ml of 10% solution of deoxycholate was added to the suspension with stirring and the resulting mixture was stirred at room temperature for 40 minutes. The mixture is then centrifuged to extract the precipitated fractions and removal of the greater part of the impurity proteins, cellular components, etc., the precipitate suspended in 4 ml of water and centrifuged to extract the precipitated fractions containing h6(1-163). Thus obtained precipitated fraction suspended in 3.8 ml of water and to this suspension with stirring was added 0.2 ml of 1 M Tris buffer (pH 8) and 1 ml of 10% N-lauroylsarcosinate, followed by stirring for 20 minutes at room temperature for Rast is 20 hours) at room temperature. The resulting solution was centrifuged to extract supernatant fractions containing h6(1-163) and a portion of 5 ml of this supernatant was mixed with 5 ml of water and 10 ml of 20 mm Tris buffer (pH of 7.7) and then 2.6 g of ion-exchange resin, 20-50 mesh Dowex 1-x4 chloride form, followed by stirring for 90 minutes. This ion-exchange resin was removed using a glass filter to extract readsorbing resin fraction, which is then centrifuged to extract supernatant fluid. It was applied to a cation exchange column, SP Sepharose Fast Flow, balanced pre 20 mm Tris buffer (pH of 7.7), and suirable the same buffer, using a linear gradient of 0-500 mm NaC1. After thorough dialysis each buervenich fractions from this column against 1MDM cultural environment and assessing them in a test system rat CFU-MK high TPO activity (relative activity 19000000) was detected in fractions, elyuirovaniya concentration N1 approximately 100 mm. This fraction was concentrated by a factor of 1.6 (2.5 ml; protein concentration of 25 μg/ml) using ultrafiltration devices (Ultra Free CL, nominal clipping mol. mass 5000, manufactured Millipore, cat. UFC4LCC25). In the analysis of the concentrated fractions using SDS-COUNCIL presence is

TRO-active fraction obtained as described above was tested in the test, in vivo, i.e., the active fraction was injected subcutaneously 1CR to male mice (8 weeks of age), and each group consisted of 4 animals daily for 5 consecutive days at a dose of 100 µl (with General activity, 47500, as measured in the test system rat CFU-MK) 1 pet. As a control, 100 μl of 20 mm Tris buffer (pH of 7.7) containing 100 mm Na1, were injected subcutaneously in the same way. Just before the introduction and the next day after injection, blood samples were taken from the fundus of the eye to measure quantities of platelets using microcamera (F800, Toa Iyo Denshi). In the treated TRO group platelet counts increased by an average of about 1.73 times after injection in comparison with the value before the introduction and were of 1.59 times higher than in the control group with significant difference (p<0,01, Student t-test) between them. These results showed that SRAW man, produced by E. coli and obtained as described above has the ability to increase the number of platelets in vivo.

Example 55. Large-scale culture of Cho cells

Large-scale cultivation of producing SRAW man Michalovce plasmids DF202-hT-P1 in Cho cells in Example 32, was carried out as follows. The SNO cell line were cultured and were propagated in DMEM/F-12 culture medium (G1) containing 25 nm MTX and 10% FCS. After separation of the cells with trypsin solution 1107cells were inoculable in a roller bottle (ex Falcon, Falcon 3000) containing 200 ml of the same medium, and then cultured at 37oWhen the rotation speed of 1 rpm for 3 days. After 3 days of cultivation, the culture supernatant was removed by suction and sticky SNO cells were then rinsed with 100 ml of S. 200 ml of medium D/F-12 (G1), not containing either 25 nm MTX or 10% FCS, was added to the flask and the cells were cultured at 37oWhen the rotation speed of 1 rpm for 7 days. After 7 days of cultivation, the culture supernatant was recovered as the starting material for the subsequent purification procedure. The above procedure was carried out in 500 roller bottles, receiving 100 l of culture supernatant.

Example 56. Cleaning TRO person from producing TRO person SNO cell line

1) Approximately 100 l of serum-free culture supernatant obtained in Example 55 was filtered through a 0.22 μm filter, receiving the filtrate which was then concentrated by ultrafiltration device (PLTK an ode to rarm SF (Wako Pure Chemicals) and Pefabloc SC (Merck), are inhibitors of proteases, was added to the obtained aqueous solution at final concentrations of 1 mm and 0.35 mm, respectively, receiving 3628 ml fractions mol. weight of 30,000 or more (protein concentration of 1.60 mg/ml; total protein 5805 mg; relative activity 1230000 and General activity 7149000000). The fraction is then subjected to Western-blotting as described in Example 45. As a result, the presence of protein TRO was detected by mol. weight between 66000 and 100000. A more detailed study it was found that more low molecular weight species TRO than this TRO was contained in passing through the column fractions.

Ultrafiltrate containing TRO mol. weighing not more than 30,000, was concentrated separately using an ultrafiltration device (PLGC Pellicon cassette, nominal clipping mol. weight of 10,000, Millipore), receiving 1901 ml fractions of low molecular weight TRO (protein concentration of 0.36 mg/ml; total protein 684 mg; relative activity 245500; total activity 167900000). This shows that during the cultivation were established, these low molecular weight species TRO.

Then 3614 ml fractions containing TRO mol. weight of 30,000 or above, was added 764 g of ammonium sulfate and 144,5 ml containing 0.5 M sodium citrate buffer (pH 5.5), getting 4089 ml rastv the rat sodium buffer. After removing insoluble substances from this solution by centrifugation was obtained a clear solution.

This clear solution was then applied on the column Macho-Prep thyl NS (SIV-Rd, cat. 156-0080; diameter 5 cm, height of layer 24.5 cm), previously equilibrated to 20 mm citrate buffer (pH 5.5) containing 1.2 M ammonium sulfate at 25 ml/min flow rate. After the download is complete elution was performed containing 20 mm sodium citrate buffer (pH 5.5) containing 1.2 M ammonium sulfate. Elyuirovaniya fraction was then concentrated by ultrafiltration device (ex Filtron, Omega Ultrasette, nominal clipping mol. mass 8000), receiving passing through the column fraction F1 (4455 ml; protein concentration 0,400 mg/ml; total protein 1780 mg; relative activity 1831000).

Then 20 mm citrate PA (pH 6.0) as an eluting buffer was passed through the column, receiving the eluate, which was then concentrated on the same ultrafiltration device Omega Ultrasette, nominal clipping mol. mass 8000), collecting the fraction F2 (1457 ml; protein concentration 0,969 mg/ml; total protein 1411 mg; relative activity 1715000). In fraction F2 presence of a protein having the mol. mass from 66000 to 100,000, was confirmed by SDS-PAGE. Western analysis showed that h is Seeley on the column, SP Sepharose Fast Flow (Parmacia Biotech, cat. 17-0729-01; diameter 5 cm, height of layer 12 cm) equilibrated previously 20 mm PA-citrate buffer (pH 6.0) at a flow rate of 15 ml/min. After applying conducted elution of 20 mm PA-citrate buffer (pH 6.0) containing 50 mm N1. The eluate was collected and it was named fraction F1 (3007 ml; protein concentration 0,226 mg/ml; total protein 679 mg; relative activity 88830). Then eluting buffer was replaced with 20 mm PA-citrate buffer (pH 5,4) containing 750 mm N1 to collect elyuirovaniya fraction F2 (931 ml; protein concentration 0,753 mg/ml; total protein 710 mg; relative activity 5558000), which was then concentrated to 202 ml using ultrafiltration device (Filtron, Omega Ultrasette, nominal clipping mol. mass 8000; and Omicon MIND 3 membrane).

Then concentrated containing TPO activity fraction F2 (197 ml) was applied to a gel filtration column Sphacryl S-200H (Parmacia Biotech, cat. 17-0584-05; diameter of 7.5 cm, height of layer 199 cm) at a flow rate of 3 ml/min Erwerbende volumes 1200-1785 ml, 1785-2010 ml, 2010-2280 ml and 2280-3000 ml were collected separately, getting fractions F1 (585 ml; protein concentration of 1.00 mg/ml; total protein 589 mg; relative activity 4118000), F2 (225 ml; protein concentration to 0.263 mg/ml; total protein 59,2 mg; relative activity 2509000), F3 (270 is 0,0467 mg/ml; total protein of 33.6 mg; relative activity 1155000), respectively. Thus, fractionated TRO activity had a wide range of mol. mass, as determined by gel-filtration. After SDS-RADA faction with subsequent Western blot analysis it was found that F1 had TRO molecules mol. mass 66000-100000 (mostly); F2 contained TRO molecules mol. mass 32000-60000; and F3 contained TRO molecules mol. mass 32000-42000. All types of molecules TRO had TRO activity.

On the basis of the sequencing of N-terminal amino acids of the molecule TRO mol. mass 66000-100000 we confirmed that the TPO molecule had the amino acid sequence of the protein encoded by the gene SRW person.

In addition, TPO molecule having a mol. a lot 66000-100000, was subjected to experiments with enzymatic cleavage using glycosidase enzymes consisting of N-glycanase (ex Genzyme, cat. 1472-00), neuraminidase (Nakarai-Tesqu, cat. 242-29 R), endo-a-N-acetylgalactosaminidase (Seikagaku Kogyo, cat. 100453) and O-glycosidase (Boehriger Mannheim Biochemica, cat. 1347101), separately or in combination, and then SDS - PAGE analysis. In the result it was found that the polypeptide part of the TRO has a mol. the weight of approximately 36000, as expected on the basis of his tearer CLASS="ptx2">

Example 57. Cleaning TRO person from producing TRO person SNO cell line

1) To 1 l of serum-free culture supernatant of Cho cells, obtained as described in Example 55 was added 211,4 g of ammonium sulfate, after which the mixture was filtered through a 0.2 μm filter (ex Yelman Science, cat. 12992). The obtained filtrate was applied to a column Macho-Rger Methyl NS (Bio-Rad, cat. 156-0081, diameter 50 mm, height of the layer 90 mm), balanced pre-20 mm PA-acetate buffer (pH 5,6), containing 1.2 ammonium sulfate, at a flow rate of 15 ml/min. After applying conducted elution of 450 ml of 20 mm PA-acetate buffer (pH 5,6) containing 1.2 M ammonium sulfate. Then elyuirovaniya fraction was applied on the column, Vydac C4 reversed-phase (The Separation Grouh, cat. WAR, diameter 4.6 mm, height 250 mm) at a flow rate of 0.75 ml/min After washing the column with 10 mm Tris buffer (pH 6.4) containing 5% ethanol (known as the developing solvent A) within 15 minutes elution was performed within 66 minutes linear gradient from developing solvent And 10 mm Tris-buffer (pH 6.4) containing 94% ethanol (known as the developing solvent). The resulting chromatogram is shown in Fig.15. In the SDS-RADA analysis of molecules with mol. a lot 65000-any samples appeared on the gel SDS-PAGE as a single band (see Fig.16). Subsequent Western analysis showed that the resulting protein is a TRO.

Analysis of N-terminal amino acids of this protein samples found that the protein had an amino acid sequence of the protein encoded by the gene SRW person.

Example 58. The preparation of recombinant virus for the expression of TPO person in insect cells

Plasmid pHTP1 obtained in Example 30, were digested with restrictase EcoR1 and Notl and then subjected to electrophoresis in 1% agarose gel, receiving strip 1200 p. N., which was then purified using the kit for purification of DNA Prep-A-Depe. Then the purified DNA ligated with the transport vector VL 1393 (1nvitrogen), the preprocessed by the same restrictases, followed by transformation into Competent cells of the strain E. Li (Toyo Boseki). From the obtained colonies were selected clone pVL 1393/h TRO, containing the coding region of the full length cDNA TRO person. Then from this clone was obtained plasmid DNA by the method described in Molecular Cloning (Sambrook et al., Cold Spring Habor Laboratory Press, 1989), followed by transfection into cells insect Sf 21 1nvitrogen) using a set of BaculoGold transfectionTM(Farmingen). Transfetsirovannyh cells were cultured in Sf-900 with 1:109-1:105and approximately 7105Sf 21 cells were infected with 1 ml of the diluted supernatant when the 27oC for 1 hour in a Cup with a diameter of 35 mm After removal of the supernatant 1% hot agarose in Sf-900 medium was then placed into the culture and allowed to harden, after which he convetional for 6 days at 27oWith in a humid atmosphere. Only the resulting plaque was viscerale and out of the virus was released in 200 μl Sf-900 medium, Sf 21 cells were infected received viral clone in 24-hole tablet for propagation of the virus. Part of the virus-containing fluid obtained from single plaques were treated with a mixture of phenol/chloroform followed by precipitation with ethanol for isolation of viral DNA, which is then used as a matrix for holding R using primers specific for the cDNA TRO person. The recombinant virus containing the cDNA TRO person, selected on the basis of amplification of the specific DNA fragment. Then Sf 21 cells were infected with supernatant containing the recombinant virus carrying the cDNA TRO person, for the propagation of the virus.

Example 59. The expression of TPO person in Sf 21 insect cells and identification of TPO activity

Sf 21 cells kultivirovalos, bearing cDNA TRO person obtained in Example 58, 27oC for 1 hour, after which the cells were cultured in Sf-900 medium at 27oC for 4 days to obtain a culture supernatant. The obtained culture supernatant was subjected to a changing environment 1MDM culture medium on column NTM-5 (Pharmacia). Significant TPO activity in the received supernatant was found in a dose-dependent as in the test with rat FU-MK and M-e-test. Recombinant TPO person, expressed in Sf 21 cells, identified by Western analysis as described in Example 45.

Example 60. Laying chain variant TRO person, h6(1-163) derived from clone pCF536/h6(1-163), carrying the sequence of bases TRO person, by its expression in E. coli, using N-lauroylsarcosinate of sodium and sulphate of copper and cleaning h6(1-163)

30 g of frozen produce h6(1-163) recombinant microorganism obtained in Example 43, suspended in 300 ml of water, was destroyed in a disintegrator high-pressure 10000 psi, Rannie High Pressure Laboratory) and then centrifuged, removing the precipitated fraction. This fraction of sediment suspended in 90 ml of water and with stirring was added water up to about the% of deoxycholate sodium with stirring, followed by stirring for 30 minutes at room temperature. The precipitated fraction was recovered by centrifugation and removed most of the impurity proteins and components of microorganisms. To this precipitate was added 180 ml of 5 mm DDT, receiving the suspension, which is then centrifuged, collecting the fraction of sediment containing h6(1-163). To the precipitate obtained was added 300 ml of water, getting a suspension, to which was then added water to the amount of fluid 570 ml with stirring. 30 ml of 1 M Tris buffer (pH 8), 150 ml of 10% N-lauroylsarcosine was added to this mixture at room temperature for 20 min stirring to dissolve h6(1-163). To the solution was added to 750 μl of 1% copper sulfate, and the mixture was stirred overnight (about 20 hours) at room temperature. After removing the supernatant fraction containing h6(1-163), by centrifugation to 750 ml of this supernatant was added 750 ml of water and 1500 ml of 20 mm Tris buffer (pH of 7.7), followed by adding 3 ml of Polysorbate 80 (Nikko Chemicals) under stirring. To the resulting solution were added 600 g of ion-exchange resin Dowex 1-X4 (20-50 mesh, chloride form) and stirred for 90 minutes at room temperature. After removing readsorbing fractions using a glass filter, ion exchange resin was washed with 750 ml of 20 mm Tris buffer (pH of 7.7). The combined solution readsorbing is Alonso Q Sepharose Fast Flow (1D 5 cm x 10 cm), balanced 20 mm Tris buffer (pH of 9.2) containing 0.1% Polysorbate 80, to extract readsorbing faction. the pH obtained readsorbing faction brought to 7.2 with hydrochloric acid, was applied to a cation exchange column, Sp Sepharose Fast Flow (1D 5 cm x 10 cm), equilibrated to 20 mm Tris-buffer (pH of 7.2) containing 0.1% Polysorbate 80, and then suirable linear gradient from 0 to 500 M N1 in the same buffer. Erwerbende fractions were subjected to SDS-RADA analysis to collect fractions of the solid to which was then added triperoxonane acid up to a concentration of 0.1%. After applying the resulting solution on the column, Capcell Pak 5 μm, 300A Cl (1D 2.1 cm x 5 cm x 2, Shiseido) was performed L with reversed phase according to the method of elution linear gradient with increasing concentration of 1-propanol in 0.1% triperoxonane acid. The eluate was analyzed by SDS-RADA in the absence of reducing agent. The result was fractionally three fractions on the basis of the provisions of the elution in HPLC with reversed phase and each fraction was diluted 3 times with 0.1% triperoxonane acid. Each diluted fraction was applied to a column HPL with reversed phase in the manner described. Obtained three fractions TRO were designated as Fr. S-a, Fr. S-b and Fr. S-c, depending on the order of elution on L from obroshennoye detected by mol. the weight of approximately 18 kDa (Fr. S-a), approximately 19 kDa (Fr. s-b) or 18 kDa (Fr. S-C) (see Fig.18). After their amino acid analysis of each of the amino acid compositions were almost the same with the corresponding value determined from the information sequence. Additionally, the results of N-terminal amino acid analysis showed that were obtained in the expected sequence. The amount of protein in each fraction, a certain amino acid analysis, was 0.64 g (Fr.S-a), is 1.81 mg (Fr. S-b) and to 3.49 mg (Fr. S-C). After a full dialysis of each fraction against 1D medium was M-e test. In the relative activity of TPO activity in each fraction was about 1620000 (Fr. S-a), 23500000 (Fr. S-b) and 746000000 (Fr. S-C).

Example 61. Laying chain variant TRO person, h6(1-163) derived from clone F536/ /h6, carrying the sequence of bases TRO, through its expression in E. Li, using guanidinate and cysteine-cystine and cleaning h6(1-163)

50 g of producing h6(1-163) frozen recombinant microorganism obtained in Example 43 was added to 500 ml of water and suspended, was destroyed using devices high pressure (10000 si, Rannie High Pressure Laboratory) and then centrifuged to extract the fraction of sediment. FRAM water. To the suspension was added 15 ml of 1 M Tris buffer (pH 9,2), 900 μl of 1 M DTT, 3 ml of 0.5 M EDTA and 30 ml of 10% of deoxycholate sodium and stirred at room temperature for 30 minutes. After centrifugation were extracted fraction of the sediment, leaving the supernatant large part of the impurity proteins, components of the microorganism, etc. To the precipitate was added 300 ml of 5 mm DTT and suspended, after which the precipitated fraction containing h6(1-163), collected by centrifugation. The obtained precipitated fraction containing h6(1-163), suspended in water in a total volume of 104 ml of the suspension was added 20 ml of 1 M Tris buffer (pH 8.5) and then 376 ml of 8 M handinhand with stirring and then stirred at room temperature for 10 minutes to dissolve h6(1-163). To the solution was added 2500 ml of 20 mm Tris buffer (pH 8.5) containing 0.1% Polysorbate 80 and then 2000 ml of 20 mm Tris buffer (pH 8.5) containing 1 M guanidinium, under stirring, followed by adding 5 mm cysteine and 0.5 mm cystine. Thus obtained solution was incubated overnight at 4oC and then centrifuged, the receiving h6(1-163) in the supernatant, which was then concentrated using ultrafiltration membrane Prep Scale UF cartridge PLDC (Millipore). The buffer of this concentrate was replaced with 20 mm Seeley on anion-exchange column of Q Sepharose Fast Flow (1D 5 cm x 10 cm), balanced 20 mm Tris buffer (pH of 9.2) containing 0.1% Polysorbate 80, and then suirable linear gradient from 0 to 500 M N1 in the same buffer, receiving a fraction (240 ml), elyuirovaniya at approximately 20-150 mm N1. The obtained fraction was diluted 4 times in 20 mm Tris buffer (pH 7,2), bringing the final volume up to 960 ml, and then brought the pH to 7.2 with acetic acid, was applied to a cation exchange column, SP Sepharose Fast Flow (1D 5 cm x 10 cm), equilibrated to 20 mm Tris-buffer (pH of 7.2) containing 0.1% Polysorbate 80, and then suirable linear gradient from 0 to 500 M N1 in the same buffer. The eluate was analyzed by SDS-RADA, collecting the solid fraction. Fraction TRO added triperoxonane acid to a final concentration of 0.1%. The resulting solution was applied on the column Capce11 Pak 5 μm, 300A C1 (1D 2.1 cm x 5 cm x 2, Shiseido), then spent L with reversed phase using the method of linear elution gradient with increasing concentration of 1-propanol in 0.1% triperoxonane acid. The eluate was analyzed by SDS-PAGE in the absence of a reducing agent and fractionally into two fractions depending on the position of elution HPL with reversed phase. Two factions TRO were designated as Fr. G-a and Fr. G-d based on the order of their elution L with reversed phase (see Fig.17). Each is a band at mol. the weight of approximately 18 kDa (Fr. G-a), or approximately 32 kDa (Fr. G-d) (cm. Fig.18). Further SDS-RADA analysis of these fractions in the presence of a reducing agent showed that in both fractions was detected a band at mol. the weight of approximately 20 kDa. The result of amino acid analysis of these fractions showed that each of the amino acid compositions nearly identical to the corresponding theoretical value determined from the information sequence. Additionally, the results of N-terminal amino acid analysis showed that these fractions contained the expected sequence. The amount of protein of each fraction determined from the results of amino acid analysis, was of 2.56 mg (Fr. G-a) or of 1.16 mg (Fr. G-d ). After a full dialysis each fraction against 1MDM medium was M-e test. As a result, this fraction had a relative activity SRW approximately 3960000 (Fr. G-a), or approximately 7760000 (Fr. G-d).

Example 62. Construction of recombinant vector DEF202-hO163 for the expression of TPO person partial length (amino acids 1-163) (hereinafter called hO163) inside the SNO cells

Vector pDEF202 constructed in Example 31 was treated with restrictase R1 and S1 retrieve large vector fra cDNA hO163, which was obtained by processing the plasmid F18S-hO163 containing cDNA ATRO, encoding amino-21-163 (SEQ 1D 13), restrictase EcoR1 and Spe1, receiving expressing vector pDEF202-hTPO163. This plasmid contained the beginning of replication (origin) SV 40, 1--the promoter of the elongation factor of the person, the site early polyadenylation SV 40, mouse DHFR of minigun, start replication pUC18 and gene-lactamase (AMRr), in which cDNA hO163 connected to the website in the direction 5'-3' 1--the promoter of the elongation factor of the person.

Example 63. Expression hO163 in Cho cells

The SNO cell line (dhfr-strain, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77, p. 4216, 1980) were grown in minimal basic medium (-MEM(-) with thymidine and gipoksantin) containing 10% fetal calf serum, in a Cup 6 cm (Faleon) and then transformed with plasmid pDEF202-hTPO163 by way of transfactual (Seikaga Ku Kogyo K. K.).

Briefly, 20 µg of plasmid DF202-hO163 obtained in Example 62, was mixed with 240 μl of 0.3 M NaC1, then with a mixture of 20 μl of transfactual and 220 ál of water. This DNA solution was added dropwise to the Cup and were cultured for 6 hours in CO2-incubator. The medium was removed from the Cup, which was then washed twice MEME(-) was added containing 10% DMS O-MEM(-) before incubation for 2 minutes in to the(-) gipoksantin and thymidine) were cultured for 2 more days, then there was a selection containing 10% cialisbuynow FCS selective medium (-MEM(-) without gipoksantina and thymidine). The selection was performed by trypsinization cells, distributed on 5 cups 10 cm or 20 24-hole cups 1 Cup (6 cm) and continued cultivation with change of medium every 2 days. Supernatant from cups or holes tested on TPO activity using BA/F3-test, observing the activity of TFS. The cells, which was observed TPO activity in the culture supernatant, transferred to a fresh Cup or fresh holes after separation to a cell concentration of 1:15 selective medium containing 25 nm methotrexate. The cells are then rekultivirovana for growth and cloning of cells resistant to methotrexate.

Alternatively, the transformation of Cho cells can be cotransfected F18S-hO163 and G1 in Cho cells.

SNO strain (SNO-DU11), transfetsirovannyh the plasmid pDEF202-hTPO163, was deposited by the author of this application January 31, 1995 under accession FERM BP-4989 at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 64. Large-scale culture of Cho cells

Large scale cultivation of producing hO163 Cho cell line (Cho 109 is xantina and thymidine] ), which was obtained by transfection of expressing hO163 plasmids pDEF202-hTPO163 in Cho cells in Example 63, was carried out as follows. SNO 109 cells were grown in DMEM/F-12 medium (G1) with 10% FCS. After collecting the cells (or trypsinization) using trypsin solution 10 million cells were inoculable in a roller bottle FALCON 3000) containing 200 ml of the same medium, and cultivated at a speed of 1 rpm, at 37oWith in 3 days. Culture medium was removed by suction and the surface of the cell cultures were rinsed with 100 ml of S. Then the cell culture was added to 200 ml of DMEM/F-12 (G1BCO) without 10% FCS and cultured for 7 days at 37oWith about 1/min. of Culture supernatant was collected and used as source material for the next stage of purification. Similar procedures were carried out in 300 roller bottles, receiving 60 l of serum-free culture supernatant.

Example 65. Cleaning CTRO of producing hO163 SNO cell line

1) 60 l of serum-free culture supernatant obtained in Example 64, filtered, collecting the filtrate which was then concentrated using ultrafiltration device (Filtron, clipping mol. weight 10000), receiving concentrated is in peptide T was shown by the presence of protein h163, expressed in mol range. weight from 20,000 to 26000.

The concentrated culture supernatant (537 ml) was treated on a column of Sephadex G-25 Fine (Pharmacia Biotech, cat. 17-0032-02; diameter 10 cm and height 30 cm), pribramnamesti 10 mm PA-phosphate buffer (pH 6.8), getting protein fraction F1 (938 ml, a protein concentration of 4.9 mg/ml, total protein 4594 mg) in solution in 10 mm PA-phosphate buffer (pH 6.8).

This protein fraction (929 ml) column of Sephadex G-25 Fine was applied at a flow rate of 15 ml/min to a column of SP Sepharose Fast Flow (Pharmacia-Biotech, cat. 17-0729-01; diameter 5 cm and height 12 cm), equilibrated previously 10 mm PA-phosphate buffer (pH 6.8), followed by elution of 10 mm PA-phosphate buffer (pH 6.8), then with the same buffer containing 10% ethanol. Eluate were United in the form of fraction F1 (1608 ml, a protein concentration of 2.13 mg/ml, total protein 3426 mg). Then there was a second elution of 10 mm PA-phosphate buffer (pH 6.8) containing 750 mm N1 and 25% ethanol, collecting the main eluate hO163 fraction F2 (651 ml, protein concentration 1.67 mg/ml, total protein 1087 mg).

It contains TRO activity fraction F2 (200 ml) column of SP Sepharose Fast Flow was added ethanol and purified water, receiving 300 ml of 45% (the final. conc.) ethanol solution. Insoluble substances, obrazowe the 17-0727-02; diameter 2 cm, height 20 cm), equilibrated previously 50% solvent A (10 mm PA-acetate buffer, pH 6,7) plus 50% solvent B (10 mm PA-acetate buffer, pH of 6.7, containing 90% ethanol) at a flow rate of 2 ml/min and Then the column was washed 55% solvent a plus 45% solvent to remove readsorbing substances. Thereafter, elution was performed at a flow rate of 1.5 ml/min in accordance with the following elution profile; 50% b for 5 minutes; linear gradient from 50% to 100% In over 140 minutes and then 100% b for 35 minutes. Fractions were collected at intervals of 5 minutes (corresponding to 7.5 ml volume). All fractions were subjected to SDS-RADA and then Western analysis to test the range of elution hO163. In the result it was found that highly hO163 with an average mol. weight of from about 20,000 to 26000 eluted in the range 66-87% ethanol. Among these proteins h 163 protein hO163 mol. weight was higher blueraven earlier from the column with reversed phase, suggesting that the glycosylated molecule hO163 has a high hydrophilicity.

To 88,8 ml elyuirovaniya faction hO163 (i.e., fraction hO163 (90 ml), elyuirovaniya at a concentration range of 68-86,5% ethanol) from the column SOUR CE 15RPC added S, and this with This concentrate was then applied to a column of Superdex 75 rd (Pharmacia-Biotech, cat. 17-1070-01; the diameter of 2.6 cm and height 60 cm), equilibrated in advance with 10 mm PA-phosphate buffer (pH 6.8) containing 10% ethanol, flow rate 1.5 ml/min from time of 60 minutes after application, erwerbende fractions were collected in 6 ml each (i.e., every 4 min). In the hO163 was elyuirovaniya in fractions of numbers of tubes 16-31, as determined using SDS-PAGE. This position corresponds to the elution range of molecular weight approximately 44000 to about 6000, as determined by gel-chromatography using standard markers, mol. mass (a mixture of Bio-Rad Gel Filtration Standard, cat. 151-1901; and Calbiochem insulin, cat. 407696). Additionally, these molecules hO163, apparently, suiryudan in order of decreasing degree of glycosylation. All types hO163 tubes 16-31 (eluting volume 180-276) was collected as fractions FA. In addition, the fraction of test tubes with numbers 16-18 (eluting volume 180-198 ml), 19-24 (eluting volume 198-234 ml) and 25-31 (eluting volume 234-276 ml) were collected separately and designated as fraction FH, FM and FL, respectively. Also the fraction FA was obtained by combining parts of the fractions FN, FM and FL. This is shown in Fig.19.

2) Then faction hO163 FN, FM, FL and F from the column Superdex 75 rd set forth in (1) will be illustrated in more detail, the N-terminal aminian in Example 1, but most of the N-terminal residues Ser could not identify. This suggests that O-linked sugar added to the N-terminal Ser. Further, it was confirmed that the sequence after N-terminal Ser is the amino acid sequence expected on the basis of gene sequence h. Protein concentrations hO163 in FH, FM, FL and FA were to 10.2 ng/ml and 6.2 ng/ml, from 0.84 ng/ml 3.2 ng/ml, as determined by amino acid analysis (cq. TAQ method, Wasters), respectively, provided that these concentrations were concentrations of the peptide parts of molecules that do not contain sugar chains. These fractions (100 ng each) were subjected to SDS-RADA when non conditions using Multigas 15/25 (Dai-ichi Depending Jakuhin, 15-25% preformed polyacrylamide gel) or reducing conditions using DTT, followed by staining with silver (Daiichi Pure Chemicals). In the result it was found that each of these fractions contains high-purity hO163. Average mol. mass hO163 in FN, FM, FL and FA, as calculated using DPC111 markers, mol. mass (Daiichi Pure Chemicals) as a standard when reducing conditions were 24000-21500, 23000-21000, 23000-20500 and 23500-20500, respectively (see Fig.20).

In addition, average mol. mass h in FH, 19) under reducing conditions in Western analysis, were 26000-22000, 25500-22000, 26000-21000 and 26000-21000, respectively. Heterogeneity among mol. masses FN, FM, FL and FA may be due to heterogeneity of O-United sugar chains. Therefore, each faction FN, FM, FL and FA were digested with neuraminidase (Neuraminidase, Nacala tesque, cat. 242-29 R), restored DTT and then analyzed on SDS-RADA. The result is that in all fractions average mol. the mass was about 19000, suggesting that heterogeneity mol. mass h mainly due to the heterogeneity in the number of sialic acid in the sugar chain-related protein hO163, and that hO163, thus obtained, is expressed in the form of glycoproteins in Cho cells.

3) Fractions FN, FM, FL and FA obtained in (2), were analyzed for their activity in vitro using M-e-test systems. In the relative specific activity were 511000000, 775000000, 1150000000 and 715000000/mg protein hTO163 on the mass of the peptide not containing the mass of sugar).

Example 66. Construction of the E. coli vector for the expression of TPO human (amino acids 1-332), which added a Lys at position -1 and the Met at position -2, respectively (hereinafter referred to as h (1-332), and expression hMKT(l-332)

For expression of full amino acid sequence TRO person in E. Li code.

Synthetic oligonucleotides: 21 and 22; 23 and 24; 25 and 26; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; 37, 38 or 39 and 40, were fosforilirovanii in a solution of 0.1 mm ATP, 10 mm Tris-acetate, 10 mm MD-acetate, 50 mm K-acetate using T4 kinase (Pharmacia) in the same test tube. Then 1/10 volume of a solution of 100 mm Tris-model HC1 (pH 7.5), 100 mm MgCl2, 500 mm N1 was added to the reaction mixture, boiled for 3 minutes in a water bath and allowed to cool, getting double-stranded DNA. 4 sets of double-stranded DNA, i.e., oligonucleotides 21 22/23 and 24 (combination); oligonucleotides 25 and 26/27 and 28 (combination); oligonucleotides 31 32/33 and 34 (combination With) and oligonucleotides 35 36/37 and 38/39 and 40 (combination D) separately ligated with a set for ligating DNA (Takagi-Shuso) and then the combination-And ligated together with the combination and the combination 2 combination-D, respectively receiving ligation-1, superior-2. With the use of ligation products 1 and 2 as matrices PCR was performed, in which the primers used oligonucleotides 41 and 42 for product 1 and ligating the oligonucleotides -43 and -44 to the product of ligation of 2. Products R 1 and 2 were digested S1 and EcoRV and EcoRV and Hind 111, respectively, followed by electrophoresis on 2% agarose gel and purified using a kit for treatment of amenta was subcloned into pBluescript 11 Sk + (Stratagene), split pre S1 and Hind 111 (E. coli DN used as a master). From the resulting colonies was selected clone with the sequence of bases shown in table. 6, by sequencing, and was named pBL(SH)(174-332) (see SQ 1D 154).

Further, pBL(XH)h 6T (1-163) obtained in Example 42 was subjected R as a matrix in the presence of the following synthetic oligonucleotides 45 and 46 as primers and the product R was rascally BamH1 and Sac1, followed by electrophoresis on 6% polyacrylamide gel, receiving a fragment of approximately 160 p. N. from gel.

45: 5'-GGGGGG-3' (SEQ 1D 155)

46: 5'-GGGGGGGGGG

GAGGTACGAGACGGAACAGCAGTGGTTGG-3' (SEQ 1D 156)

Also pBL(SH) (174-332) were digested S1 and Hind 111 and the cleavage product was purified using the kit for purification of DNA Rger-And-Depe, receiving a fragment of approximately 480 p. N. Obtained two fragments were subcloned into the pBluscript 11SK+(Stratagene), split pre-BamH1 and Hind 111 (E. coli DH5 was used as the host).

From the obtained clones by sequencing was selected clone with the sequence of bases shown in table. 7, and is called pBL(BH) (123-332) (see SEQ 1D 157).

pBL (XH)h6T(l-163) obtained in Example 42 was BamH1 digested and ind ago these two fragments ligated together, getting a clone pBL(XH)h6T(1-334). Next, pCFM536/h MKT (1-163) obtained in Example 52, was Xba1 digested and Sfi1, receiving a fragment of approximately 270 p. N. , which is then ligated with pBL (HN)-h6(1-334), split the same restrictases, getting a clone L (HN)h(1-334). After splitting BL (HN)h (1-334) Xba1 and ind 111 was extracted and purified fragment of approximately 1040 p. N., which encodes TRO human mutated type (amino acids 1-332). It has cloned in F536 (EP-A-136490), rescaling pre b1 and ind 111 (E. coli jM109, pre-transformed pMW1 (ATCC 39933), was used as the host). The resulting clone was named FM536/h(1-332) and an E. coli strain that carries this expressroute vector, was used as transformants for the expression of protein h(1-332) mutated type. This expressing plasmid F536/h MKT(1-332) contains the DNA sequence shown in SEQ 1D 14.

This transformant was cultured in 60 ml of LB medium containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline, overnight at 30oWith this culture (25 ml) was then added to 1000 ml of LB medium containing 50 μg/ml ampicillin, and shaking culture at the 36oWith until D600he reached 1,0-1,2. Then, after adding to the culture of approximately 330 ml LB medium at 42oWith to induce the expression of variant TRO human protein h(1-332), also known as [Met-2, Lys-1] TRO(1-332).

The resulting culture was immediately subjected to SDS-RADA analysis. It was used Multigel 15/25 Dai-ichi Chemical Co.). After electrophoresis and staining of Kumasi blue was discovered protein that is specific for the induction of gene expression, mol. the weight of approximately 35 kDa on the gel in respect of transformant. This specific protein confirmed the expression of protein hMKT(1-332). In addition, after SDS-RADA electrophoresis and electroblotting (using intracellular membrane) expressed protein reacted with antibodies against peptide NT obtained in Example 45. After staining detected a band at mol. the weight of approximately 35 KD, showing that was expressed hM(1-332).

Example 67. Getting substituted derivatives TRO person, their expression in OS 7 cells and the identification of their activity

In accordance with the following procedures investigated whether TPO activity of some derivatives in which amino acids TRO person were partially replaced by other amino acids.

Vector S201 for expression in animal cells, for use When the mouse erythropoietin receptor (obtained from Dr. D'andrea in Dana Farbor Cancer Institute; Cell: 57, 277-285 (1989)), were treated with restrictase Kpn1 and EcoR1 and subjected to electrophoresis on agarose gel, receiving rkhm vector fragment which has been removed cDNA of murine erythropoietin receptor. Then using a DNA synthesizer (AU) synthesized two of the oligonucleotide to the introduction of various restriction enzymes cut sites in expressing vector. Synthesized oligonucleotides have the following sequences presented in table.IX.

These two oligonucleotide were mixed and annealed, getting double-stranded oligonucleotide, which is ligated with obtained as described above, the vector rkhm in the presence of T4 DNA ligase (Takara-Shuso), receiving expressing vector DM201. To remove the mouse DHFR to the DNA contained in pDMT201, pDMT201 vectors D201 and pEF18S was separately digested Not 1 and Hpa1 and subsequent agarose gel electrophoresis, receiving a large fragment of DNA vector D201 and a smaller fragment of the DNA vector pEF18S. Then these fragments are ligated together using T4 DNA ligase (Takara-Shuso), receiving expressing vector DMT201. This vector, as shown in Fig.21, has the origin (beginning) of the SV40 replication, enhancer sequence, main sequence late promoter of adenovirus, sostojavwegosja site early SV40 polyadenylation, the sequence of the gene VA RNA of adenovirus, early replication U18 and gene-lactamase (AMRr), together with the following restriction sites for ligation of the desired gene: Bgl 11, Pst1, Kpn 1, Xho1, EcoR1, Sma1, Spe1 and Not1. pHTP, illustrated in Example 30, the bearing cDNA SRAW man full length, split EcoR1 and S1, receiving a fragment of the cDNA of full length TPO man, who then was subcloned into the vector S201, split in advance also, getting plasmid pSMT201-hTPO.

Using the thus obtained plasmid pSMT201-hTPO as the matrix was first constructed a plasmid GL-TRO/Lu, which was used as a matrix to obtain the target derivatives, as follows.

In GL-TRO/Lue tried to hold adding leader sequence of the rabbit-globin 5'-noncoding site h by way Annweiler (Annweiler et al. , Nucleic Acids Research, 19: 3750, 1991). For this purpose, chemically synthesized two chains of DNA are presented in table.X.

SEQ 1D 160 and 161 ligated with a fragment obtained by cleavage of the vector Bluescript 11SK+ (Toyo boseki) EcoR1 and Sm1, receiving the vector GL/pBLue, in which was embedded a leader sequence.

PCR was performed using the following primernih sequences:
'
(SEQ 1D 163) (i.e., the antisense sequence 1140-1160 SEQ 1D 7, which were connected by a sequence S1 and Hind 111).

R was performed using 1 ng DNA plasmids S201-h as a matrix with 10 μm of these synthetic primers. Using set for PCR amplification Takara (Takara-Shuzo) and Programmable Thermal Controller (MJ Research) PCR was performed in a volume of 100 μl under the following conditions: 3 cycles of 94o1 min, 55o2 min and 72o2 min (cycle); followed by 20 cycles of: 94oWith 45, 55o1 min and 72o1 min (cycle). The PCR product was extracted with chloroform and then precipitated with ethanol twice, followed by suspendirovanie in 100 ál of TE buffer.

Then the PCR product was digested with restrictase Sm1 and Hind 111, were extracted with a mixture of phenol/chloroform and precipitated with ethanol. The precipitate was dissolved in 10 μl of TE buffer, after which it was subcloned into the vector GL /lue, pre-split the same restrictases (Competent strain of E. coli J109 (Toyo boseki) was used as a master). From the obtained transformed cells were selected 20 clones of plasmid DNA, essentially as described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). Purified rminater Cycle (Applied Biosystems, Inc.) and DNA sequencing machine A (Applied Biosystems, Inc.). In the result, it was confirmed that a plasmid encoding GL-TPO/pBLue had expected cDNA sequence TRO without any substitutions along its entire length. Then after splitting GL-TPO/pBLue Bg111 and Spe1 the cleavage product was extracted with a mixture of phenol/chloroform and precipitated with ethanol. The precipitate was dissolved in 10 μl of TE buffer and was subcloned into the vector pSMT201, which was cleaved by the same enzymes (Competent High E. coli JM109 (Toyo boseki) was used as a master). In accordance with a method described in Molecular Cloning (Sambrook et al., Supra), from cells transformant received plasmid DNA. Received expressing vector pSMT/GL-TRO had a structure in which the leader sequence-globin and cDNA SRW people were connected at the restriction site Bg111/Spe1 in the direction 5'-3' from the signal sequence of the splicing vector S201, as shown in Fig. 21. This vector pST/GL-TPO was used for transfection in 0S 7 cells.

To obtain substituted derivatives TRO person used PCR, in which was used the method of Ito (Ito et al., Gene, 102: 67-70, 1991). In particular, received two derivatives TRO person, in which Arg-25 and His-33 TRO person were replaced Sn and Thr, respectively. These derivatives m T7: 5'-GGGGG-3' (SEQ 1D 164) (corresponding to the area of the T7 promoter BLuescript 11 S+); Bgl11: 5'-GTG-3' (SEQ 1D 165) (for replacement sequence recognition Bgl 11); (end): 5'-GGGGGG-3' (SEQ 1D 166) (antisense sequence 1140-1160 SQ 1D 7, which are connected in sequence S1 and ind 111); 3: 5'-GGGGGGGGGGGGGGG-3' (SEQ 1D 167) (Arg 25 --> Asn SEQ 1D 7); and 09: 5'-GAGGGGGGTGGG-3' (SEQ 1D 168) (is - 33 --> Thr SEQ 1D 7).

The first stage PCR was performed using 1 ng of plasmid DNA GL-TPO/pBLue as a matrix with 10 μg of each of the synthetic primers. Combinations of primers were as follows: [1] Bgl11 and "end" primers; [2] T7 and N3; and [3] T7 and 09. Using set for PCR amplification (Takagi Shuso) and Programmable temperature controller (Mu Research) PCR was performed in a volume of 100 μl under the following conditions: 3 cycles of 94o1 min, 55o2 min and 72o2 min (1 cycle); then 17 cycles of 94oWith 45, 55o1 min and 72o2 min (1 cycle). Each of the PCR products were extracted with chloroform and precipitated twice with ethanol and the precipitate was dissolved in 100 µl TE buffer. The resulting PCR products (1 μl each) were then subjected to the second PCR. Combinations of matrices were as follows: PCR products[1] /[2] and the PCR products [1]/[3]. As primers in both cases used a combination of T7 and "end". After innumerous 1 min, 55o2 min and 72o2 min (cycle); then 9 cycles of 94oWith 45, 55o1 min and 72o1 min (cycle). Each of the obtained PCR products were added 1 μl of proteinase K (5 mg/ml), 2 ál of 0.5 M EDTA and 2 μl of 20% SDS, incubated at 37oC for 30 minutes to inactivate Taq, were extracted with a mixture of phenol/chloroform and precipitated with ethanol. After that, the precipitate was dissolved in 20 µl of sterile water and were digested gl11 and S1 followed by extraction with a mixture of phenol/chloroform and precipitation with ethanol. Thus obtained precipitate was dissolved in 10 μl of TE buffer and then was subcloned into expressing vector S201, pre-split the same restrictases, and was treated with alkaline phosphatase calf intestine (Boehringer-Mannheim) (Competent High E. coli J109 was used as a master). From the obtained transformed cells in each case were selected two clones, of which plasmide DNA was obtained essentially as described in Molecular Cloning (Sambrook et al., Supra). Purified plasmid DNA sequenced using DNA sequencing machine A and sequencing q Dye DeoxyTMTerminate Cycle (both Applied Biosystems, Inc.).

A plasmid obtained by means of PCR using these primers [1] and [3] , contained a cDNA encoding a substituted proizvodstvennonauchnoe sequence TSIGYPYDVPDYAGVHHHHHH (SQ 1D 169) were confirmed. This derivative is called [Thr33, Thr333Ser334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr344Ala345, Gly346, Val347, His348, His349, His350, His351,His352, His353] SRW.

On the other hand, a plasmid obtained by means of PCR using primers [1] and [2], contained a cDNA encoding a substituted derivative TRO (N3/SRW). Were confirmed by replacement of amino acids AGD 25(AG) to Asn(AAC) and Glu 231 (GAA) to Lys (AAA), and the lengthening of the initial-end (Gly 332) addition of the amino acid sequence S1GDVD YGV. This derivative is called [Asn25, Lys231, Thr333, Sr334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr344Ala345, Gly346, Val347, His348, His349, His350, His351, His352, His353] SRW.

Transfection in OS 7 cells of each of the obtained clone was conducted by DEAE-dextranomer way capable of handling chloroquine, as described in Example 35. Briefly, 40 μg of plasmid DNA used in transfection and after 5 days was recovered culture supernatant, which is then is LTTE in the culture supernatant COS 7 cells in which were respectively transliterowany plasmid containing cDNA encoding derivatives TRO person N3/TRO and 09/TRO, was discovered TPO activity in a dose-dependent (see Fig.22).

Example 68. Receiving insertion or deletion derivatives TRO person, their expression in COS 7 cells and identification of TPO activity

This Example shows whether TPO activity insertion and deletion derivatives hO163.

We have received the following derivatives: His 33-deletion derivative ([is33] TRO (1-163), dH33); Gly116-deletion derivative ([Gly116] TPO(1-163), dG 116); AGD 117-deletion derivative ([AGD117] TPO(1-163), dR 116); Thr-insertion derivative ([His33, Thr33The EMG34] TPO(1-163), T33'); l-insertion derivative ([His33Ala33'The EMG34] TRO (1-163), A33') and Gly-insertion derivative ([His33, Gly33'Pro34Ser38] TPO(1-163), G33'), in which hr, l and Gly were independently built between is 33 and RHS 34; and sn-insertion derivative ([Gly116, sn116', Arg117] TPO(1-163), # 116'). Ala-insertion derivative ([Gly116, l116', AGD117] TPO(1-163), A') and Gly-insertion derivative ([Gly116, Gly116', Arg117] TRO (1-163, G116'), in which Asn, Ala and Gly were ¾ derivatives T33' and N116 intended for the introduction of chain-type mucin and sugar chain-type Asn-binding chain, respectively.

The above derivative was obtained by using R according to the method described Jto et al. (Jene, 102: 67-70, 1991).

In PCR used the following primers:

dH33: 5'-GGGGGGGG-3' (SEQ 1D 172) (for His 33-deletion derived on the basis of SEQ 1D 7);

T33': 5'-GGGAGGGGGGG-3' (SQ 1D 173) (for Thr-insertion derived from hr, built between is 33 and RHS 34 in SEQ 1D 7);

A33': 5'-GGGGGGGGGG-3' (SQ 1D 174) (for l-insertion derived from Al, built between is 33 and RHS 34 in SQ 1D 7);

G33': 5'-GGGGGGGGG-3' (SEQ 1D 175) (for Gly-insertion derived from Gly, built between is 33 and RHS 34 in SEQ 1D 7);

dG116: 5'-GGGGGGGGGG-3' (SEQ 1D 176) (for Gly116-deletion derived on the basis of the sequence shown in SQ 1D 7);

dR117: 5'-GGGGGGTGTGGGGG-3' (SEQ 1D 177) (for AGD 117-deletion derived on the basis of the sequence shown in SEQ 1D 7);

# 116: 5'-GGTGGGGGGGGG-3' (SQ 1D 178) (for sn-insertion derived from sn, built between Gly116 and AGD in SEQ 1D 7);

A': 5'-GGGGGGGGGGG-3' (SEQ 1D 179) (for l-insertion derived from l, built between Gly116 and AGD 117 in SEQ settled between Gly116 and AGD in SEQ 1D 7);

dR1: 5'-GGGGGGG-3 (SEQ 1D 181) (to replace a sequence of recognition of restrictase EcoR1);

T7: 5'-GGGGG-3'(SEQ 1D 182) (corresponding to the area of the T7 promoter Bluescript 11S K+); and Not end: 5'-GGGCGGGGGGGGGGGGTGGG-3' (SEQ 1D 183) (antisense nucleotide sequence 633-653 SEQ 1D 7, to which is added the termination codon and sequence recognition Not1).

The first stage PCR was performed using as matrix 1 ng DNA plasmids GL-TPO/pBlue obtained in Example 67, and 10 μm of the synthetic primers. The combination of primers was: [1]dR1 and end Not1 or [2], mutated T7 primers (dH33, A33', G33', T33', dG116, dR117, A116', G116' # 116'). In PCR used set for PCR amplification (Takara Shugo) and Programmable thermal controller (MJ Rescarch) and R was performed in a final volume of 100 μl. In the first PCR reaction [1] one cycle was 94o1 min, 45o2 min and 72o2 min and 3 cycles were performed followed by 17 cycles in which each cycle was 94oWith 45, 45o1 min and 72oWith 1 minute on the other hand, the first reaction of [2] was performed under the conditions: 94o1 min, 55o2 min and 72oWith 2 minutes For 3 cycles, followed by 17 cycles, each of which consisted of 94oWith 45, 55o1 min and 72o1 minutes Each which was storyli in 100 ál of TE buffer.

Then there was the second stage R using 1 µl of each of the products R [1] and [2]. Used a combination of T7 and end Not1 as primers. After incubation at 94oC for 10 minutes to each reaction mixture PC was added Takara q. The second PCR was performed under the conditions: 94o1 min, 55o2 min and 72o2 min for 3 cycles, followed 94oWith 45, 55o1 min and 72o1 min for 9 cycles. Each of the thus obtained PCR products were added 1 μl of 5 mg/ml proteinase K, 2 ál of 0.5 M EDTA and 2 μl of 20% SDS and the mixture is kept at a temperature of 37oC for 30 min to inactivate q. The PCR product was extracted with a mixture of phenol/chloroform followed by precipitation with ethanol and the precipitate was dissolved in 20 μl of sterile water. After splitting EcoR1 and Not1 solution was extracted with a mixture of phenol/chloroform and then precipitated with ethanol. The precipitate was dissolved in 10 μl of TE buffer and was subcloned into the expressing vector pEF18S, pre-split the same restrictases, and was treated with alkaline phosphatase (from calf intestine, Boehringer-Mannheim). The host cells were Competent-High E. coli M109 (Toyo Boseki). From the received transformant received plasmid DNA by the method described in Molecular Cloning (which is confirmed by using q Dye DeoxyTMTerminater Cycle set for sequoiaview (Applied Biosystems) and DNA sequencing machine A the same company. In the result, it was confirmed that all of these plasmids encoding d33, A33', T33', dG116, dR117, A', G116' # 116', had the expected sequence of the cDNA TRO without any substitutions nucleotide sequence at sites other than intended for replacement sites. Moreover, G33 watched the replacement of the base [Rho(CST) --> Sr (TCT)], other than a change in the planned site.

Transfection of each of the obtained clone in COS 7 cells were performed according to the method in Example 11. Briefly, the transfection was carried out using 10 μg of each plasmid DNA DEAE-dextranomer way capable of handling chloroquine, and after 4-5 days recovered culture supernatant, which was estimated using M-e-test systems. As a result, in each supernatant of cultures of COS 7 cells transfected with plasmids encoding amino acid insertion or deletion derivatives was discovered TPO activity in a dose-dependent (see Fig.23). In contrast, TPO activity was not detected in the supernatant of the culture of COS 7 cells expressing a plasmid which pEF18S, transfusiona them and downregulation did not contain CA

1) Six areas (shown in the table. 8), which are considered to be relatively suitable as antigens were selected from the amino acid sequence TRO human SEQ 1D 191, which was synthesized 4-chain peptide-type peptide with multiple antigens (MAP) according to the method of Tam (Proc. Natl. Acad. Sci. USA, 85, 5409-5413, 1988). Two rabbits were immunizable 8 times with 100 μg of the peptide.

Separately received single-stranded peptides by binding to cysteine residue with an end of each of the peptide regions, are shown SQ 1D 119-124 (called peptide regions of NT 1-6, respectively, each of the partial peptides corresponds 8-28, 47-52, 108-126, 172-190, 262-284 and 306-332 provisions, respectively, of the sequence SEQ 1D 191). Using them as test antigens, measured by the number of antibodies in the sera obtained from the immunized thus rabbits, using enzyme immunoassay and confirmed the increase in the number of antibodies in all tested sera. Thus, these serum called forth by the antisera.

In addition, the above-mentioned single-stranded synthetic peptide having a cysteine residue that is associated with it-end, was used as antigen for the ne of the above antigenic peptides and antibodies obtained from each rabbit received separately. Specifically, antibodies against NT peptide antibodies were obtained against NT-1 peptide and antibodies against NT-2 peptide from two immunized rabbits. Next, as one example illustrates the purification of antibodies against peptide NT-1.

First, 30 mg of single-chain peptide NT having a cysteine residue that is associated with it, combined with 12 ml Sulfo Link Coupling GE1 (produced by Pierce Co. , cat. 44895). More precisely, the solution of a peptide containing the antigen, combined with the gel, balanced binding buffer (50 mm Tris, 5 mm EDTA-Na, pH 8.5), 6 times larger than the volume of the gel over a period of 15 minutes. Then, after standing for 30 minutes, the gel was washed with binding buffer in an amount of 3 volumes of gel. Then the binding buffer containing 0.05 M L-cysteine-model HC1 was added to the gel at a ratio of 1 ml/ml of gel, through which untreated groups were blocked for 15 minutes. After standing for 30 minutes, the gel was washed with binding buffer 8 times a number is equal to the volume of the gel. The operation of binding was carried out at room temperature. In this way contains the antigenic region of the peptide was coupled to the gel through a covalent link is related to 1 ml of gel in antigenic column. After taking whole blood from each immunized rabbit received 78,4 ml of antisera containing antibodies against the NP-1 peptide. 76,7 ml of this antisera (protein 3620 mg) was added to antigenic column equilibrated previously with 50 mm phosphate buffer (pH 8) containing 150 mm N1 and 0.05% of sodium azide, and then the column was washed with the same buffer. In this way received 105,9 ml passing through the column fractions (protein 3680 mg). Then adsorbed fraction was suirable 0.1 M nitrate buffer (pH 3.0), which is then immediately neutralized by the addition thereto of 21.1 ml of 0.1 M carbonate buffer (pH 9,9), was concentrated by ultrafiltration (using USO membrane manufactured by Amicon Co.) and purified in 50 mm phosphate buffer (pH 8) containing 150 mm N1 and 0.05% sodium azide. So got to 11.2 ml of purified antibodies against NT-1 peptide (protein 77,7 mg) in buffer. In the same way received antibodies against NT-2 peptide (60,0 mg), antibodies against NT2-1 peptide (18,8 mg), antibodies against NT2-2 peptide (8.2 mg), and so on

Antibodies against NT3-NT peptides receive as described above.

Example 70. Detection TRO using Western analysis

1) For the evaluation of antisera, pay, partially purified from the culture supernatant of Cho cells, which was introduced in the gene encoding amino acids-21-332 SEQ 1D 6, and in which it was expressed were subjected to electrophoresis in SDS-polyacrylamide (SDS-RADA) in accordance with the usual way and then spent electroblotting on PVDF or nitrocellulose membrane. After blotting the membrane was washed in 20 mm Tris-model HC1 and 0.5 M N1 (pH 7.5) (TBS) for 5 minutes, then washed twice with 0.1% tween 20-containing S (S) for 5 minutes and was treated with a blocking agent (Block ACE; produced by Dai Nippon Pharmaceutical Co., cat. IR-B25) for 60 minutes. Antisera containing each one of the antibodies; antibody against NT-1 peptide, antibody against NT2-1 peptide, antibody against NT3-2 peptide antibody against NT-1 peptide, antibody against T-2 peptide and the antibody against NT-1 peptide was diluted solution S containing 0.05% BSA and 10% lock ACE to 1/1000 dilutions. Divorced thus antibodies used for Western analysis as the primary antibody. In particular, a sample of recombinant TPO person treated as described above were processed S a solution containing the antibody against the TRO-peptide, of 0.05% BSA and 10% Block ACE, within 60 minutes and then washed S within 5 minutes. The ZAT is 43015), as secondary antibodies, together with 0,05% S and 10% Block ACE, within 60 minutes and then washed twice in TTBS for 5 minutes. Then it was treated with a dilution of 1/5000 labeled alkaline phosphatase avidin (produced Linco Technologies Co., cat. A108), diluted with 10% Block ACE solution S for 30 minutes and then washed twice in TTBS for 5 minutes and then in TBS for 5 minutes. Then the membrane was shown with the use of the substrate for alkaline phosphatase (manufactured by Bio-Rad Co., cat. 170-6432). Western analysis was carried out at room temperature. He confirmed that the TRO person is recognized and detected by these antisera.

2) 3 mg of each antibody against NT-1 peptide, against NT-2 peptide, against NT2-1 peptide and against NT2-2 peptide, which were purified by affinity chromatography in Example 69, weighed and biotinilated linking them to the activated Biotin (NHS-LC-Biotin 11, produced by Pierce Co., cat. 21336). The same standard sample of recombinant TPO man, which was used in (1), subjected to SDS-PAGE, and then electrically was bottiroli on PVDF or nitrocellulose membrane and subjected to conventional Western analysis using each of these biotinylated antibodies as pellicena 5 minutes and then was treated with a blocking agent (Blk ACE) for 60 minutes. Then it was treated S solution containing 1 μg/ml biotinylated antibodies against TPO-peptide, 0,05% S and 10% Block ACE for 60 minutes and then washed twice S within 5 minutes. Then the membrane was treated with a dilution of 1/5000 labeled alkaline phosphatase avidin (Leinco Technologies Co., cat. A108), diluted with 10% Block ACE TTBS solution for 30 minutes and then washed twice in TTBS for 5 minutes and then S within 5 minutes. Then the membrane was shown with the use of the substrate for alkaline phosphatase (SIV-Rad Co., cat. 70-6432). Described Western analysis was carried out at room temperature. He confirmed that the TRO person is recognized and detected these purified antibodies.

Example 71. Getting speakers with antibodies against TPO-peptide and detection SRAW man

Antibodies against TPO-peptide person obtained in Example 69, was tested for recognition TRO person. These antibodies are individually bound to the chromatographic medium to obtain columns with antibodies against TPO-peptide person. As one example further illustrates the preparation of the column with the antibody against T-2 peptide.

1) Preparing a solution containing 50 mm of sodium phosphate and 0.15 M N1 the new gel (Formyl-Gellulofine, produced by Chisso Co.) at 4oC for 2 hours. Then thereto was added 1.1 ml of a solution containing 10 mg/l of a reducing agent (trimethylaminuria (TMAU), produced by Seikagaku (Kogyo K. K., cat. 680246), and the binding was continued for another 4 hours. Then, only a portion of this gel was taken from the reaction mixture by centrifugation. 10 ml of pure water was added thereto, and only a portion of the gel was removed from the mixture by centrifugation. This process was repeated 4 times to remove unreacted antibody. Then thus obtained gel was treated with 2.1 ml of blocking buffer (0.2 M Na-phosphate, 1 M ethanolamine, pH 7.0) and 1.1 ml of a solution of a reducing agent added to it, at 4oC for 2 hours, resulting in the active group in the unreacted gel were blocked. Finally, the obtained gel was washed with water, 20 mm Tris-model HC1 and 0.15 M N1 (pH 8.0) using a centrifuge. They filled the small column tube, which was washed 3 M solution of sodium thiocyanate and 0.1 m solution of glycine-model HC1 (pH 2.5), and then again balanced 20 mm Tris-model HC1 and 0.15 M N1 (pH 8.0). Balanced thus the column was kept.

Gel with antibodies against TPO-peptide column with antibodies against NT-2, the per unit volume of the gel, was 1.9 mg/ml gel. In the same way as described above, preparing the gel, which was due to 21.8 mg per ml of gel 1gG, not having TRO as antigen. This gel was used as predalone (hereinafter called precolonial with antibodies) to remove nonspecific binding of molecules from the speakers with antibodies against TPO, as mentioned in (2).

2) below is another example of the preparation of the column with antibodies against NT-2.

A standard sample of recombinant TPO man who was partially purified from the culture supernatant of Cho cells into which has been introduced a gene encoding the amino acid sequence of any of SEQ 1D 119-124, which expressively in these cells, was added to predalone with antibodies (containing 1 ml of gel) prepared in (1), and 10 times missed volume of 20 mm Tris-model HC1 (pH 8.0) and 0.15 M N1 equal to the volume of the gel through the column, collecting passing through the column fraction. Then suirable fraction adsorbed on predalone, 10 times the volume of gel acidic eluent (0.1 M glycine-model HC1, pH of 2.5). Then the fraction passing through predalone, was added to the column with antibodies against NT-2 (containing 2 ml of gel) prepared in (1), and the column was washed 10 times the volume of gel 20 mm fraction, adsorbed on the column 8 times the volume of gel is acidic (0.1 M glycine-model HC1, pH of 2.6). These fractions were analyzed using SDS-PAGE, which confirmed that the TRO is not adsorbed on predalone with antibodies, but specifically binds to the column with antibodies against NT-2, and that almost the entire SRW added to this column, the sample is contacted with the last column. Based on this experiment, it was confirmed that not less than 200-300 μg/ml of gel TRO contacted with the gel in the last column.

Example 72. The effect of TRO on the increase in platelet count

20 normal male mice of strain 1R, eight weeks of age (blood has been taken from them the ophthalmic veins and platelet counts were measured using microtiter F-800, manufactured Toa IRE Denshi) was divided bezvyhodnym way into 4 groups. One of 4 groups (control-iv group) were injected with 100 μl S intravenously (iv) once daily for 5 consecutive days. One of the other groups (SRW-iv group) were injected with 100 μl of a solution (prepared by substitution of concentrated solution TRO-active fraction F2 SP Sepharose Fast Flow, obtained in Example 56, the buffer S using column N-25 [Pharmacia Biotech; cat. 17-0852-02] with further dilution S; soteriades is the GUI group (control-SC group) were injected with 100 μl S subcutaneously (SC) 100 μl of PBS 1 time a day for 5 consecutive days, while the last group (SRW-SC group) were injected with 100 μl of 1 times a day for 5 consecutive days, as well as in a group TPO-iv, but subcutaneously (SC). After 6, 8, 10, 13 and 15 days from the beginning of the injection of blood from the ophthalmic vein was taken from each of the mice and the number of platelets counted using microcamera (F-800; Toa IRE Denshi). Changes in numbers of platelets are given in Fig.24. Thus, in the control group-iv the number of platelets increased by 44% compared with the number before the introduction of even on the 6th day (day 0 is the day of the beginning of the introduction) when platelet counts were higher, and in the control group - SC increased only 47% have been noted on the 8th day, when platelet counts were higher. On the other hand, in the group TPO-iv observed an increase of 177% on day 6 compared with quantities before the introduction and in the group SRW-Sc increase of 347% was noted on day 6 and on day 8 was observed the highest increase 493%.

These results confirmed that the TRO person increases the number of platelets regardless of differences in the type and route of administration.

Example 73. The effect of TRO on the increase in platelet count

16 normal male mice of strain SN/J 11 weeks of age (blood was taken at the Toa IRE Denshi) was divided bezvyhodnym way into 4 groups. One of the four groups (control group) were injected with 100 μl S subcutaneously 1 time a day for 5 consecutive days. Another group (group SRW-1) were subcutaneously injected with 100 μl of a solution (prepared by substitution TRO-active fractions Sephacryl S-N obtained in Example 56, the PBS buffer through the column NA-25, with further dilution S; contains the relative activity 83000 in M-e-test) 1 time a day for 5 consecutive days. Another group (group SRW-2) were injected with 100 μl of the solution (obtained by diluting TRO-active fractions, applied in the group SRW-1, S 2 times; containing the relative activity in 41500 M-e-test) subcutaneously 1 time a day for 5 consecutive days. The last group (group SRW-3) were injected with 100 μl of the solution (obtained by diluting TRO-active fractions used in the group SRW-2, even 2 times; containing relative activity 20750 collection in M-e-test) subcutaneously 1 time a day for 5 consecutive days.

Blood was taken from the orbital vein of the mice at the 6 th, 8 th, 10-th and 12-th day after injection and the number of platelets counted using microcamera (type F-800; Toa Iyo Denshi).

Changes in numbers of platelets are given in Fig.25. Thus, in the control of GRU the deposits were in amounts of platelets in all groups, moreover, higher platelet counts were on the 8-th day after injection. In addition, these groups were marked differences according to response dose-response. So, in the group SRW-1 is approximately 200% increase was noted on day 6, the magnification was increased to about 270% on day 8, and then, however, observed a reduction, even on the 12th day still observed a 65% increase, between the observed significant difference from control group (p<0,01; test Dunnet multiple comparisons). In TRO-2 group found similar increases, although the increase was lower than in group SRW-1, increase of 140% and 160% was observed on the 6th and 8th day, respectively. On the 12th day of the platelet count decreased to approximately the same level that was in the control group. Increasing action under TRO-3 was weaker than in the TRO-2, and on the 8th day, when platelet counts were higher, said 110% increase and was also a significant difference (p<0.01) compared with the control group.

The above results showed that the TRO person increases the number of platelets regardless of the strain of mice, and that its action has the feature according to response dose-response.

Example 74. the necks 1CR eight weeks of age were injected with 200 mg/kg 5 FU intravenously and shared bezvyhodnym way into 2 groups (each group consisted of 15 mice). The next day (day 1) one group (the control group) were injected with 100 μl of PBS subcutaneously 1 time a day for 5 consecutive days, while another group (group SRAW) were injected with 100 μl of TRO-active fraction used in Example 72 (containing the relative activity 211900 in M-e-test) subcutaneously 1 time a day for 5 successive days. On the 4th, 6th and 8th days 5 mice were randomly selected from each of the two groups, then took the blood from the ophthalmic veins and the number of platelets counted using microcamera (type E-2 500; Toa Iyo Denshi). Changes in numbers of platelets are given in Fig.26. Thus, in the control group the number of platelets after administration of 5-FU decreased over time and on the 6th day of this value was the lowest (approximately 28% of the value before the introduction of the 5-FU), although on the 8th day was celebrated by about 1.3-fold increase in response to this reduction. Even in the case of a group SRW platelet counts after administration of 5-FU decreased over time and on the 6th day of this value was the lowest (approximately 52% of the value before the introduction of the 5-FU). However, the difference between the quantities of platelets on the 4th and 6th days were very low and on the 6th day remained significantly high (p<0,05: Dunnet test) is before the introduction of 5-FU.

The result confirmed that the TRO person inhibits the decrease in the number of platelets caused by an anticancer agent.

Example 75. Therapeutic effects of TRO for thrombocytopenia

Hydrochloride of nimustine (ASPI) (50 mg/kg) were intravenously injected with each of the 36 male mice 1CR seven-week of age and then these mice were divided bezvyhodnym way into two groups (each group consisted of 18 mice). The next day (day 1) one group (the control group) were injected with 200 μl of PBS subcutaneously twice a day for subsequent days, while another group (group SRAW) were injected with 200 μl of TRO-active fraction used in Example 73 (without dilution PBS), to which was added 0.04% tween 80 (relative activity 380000 in M-e-test) subcutaneously twice a day for subsequent days.

After injection, mice in each group were randomly divided into three groups: one group blood was taken on the 5th and 12th days; from another group on the 8th and 14th days and the third group on the 10th day. Blood was taken from the ophthalmic veins and the number of platelets counted using microcamera (type F-800; produced by Toa Iyo Denshi). Changes in numbers of platelets are given in Fig. 27. Thus, in the control group Kolskiy (approximately 29% of the value before the introduction of CADI) and the recovery was only about 49% and 74% at the 12th and 14th days, respectively. On the other hand, in the group SRW noted a decrease to approximately 38% of the value before the introduction of ACNU on the 5th day and then the situation changed to increase. So, on the 8th day the amount was restored to approximately 63% of the value before the introduction of ACNU and on the 10th day and after platelet counts were greater than before the introduction of CADI. So, on the 12th and 14th days of the increases were approximately 300% and 400%, respectively.

As noted above, for 8-10 days, when it was noted a decrease in the control group, in the group SRW have seen an increase and 10-day platelet counts were higher quantities before the introduction of CADI. Therefore, it was confirmed that the TRO person accelerates the return to normal for thrombocytopenia caused by an anticancer agent.

Taking into account the results of Example 74, it can be expected that TRO person inhibits the decrease of platelets and accelerates the return to normal in the event of thrombocytopenia, regardless of the type of anticancer agent.

Example 76. Therapeutic effect of TRO on thrombocytopenia after bone marrow transplantation (BMT)

48 male mice of strain SN/N seven-week of age were irradiated 10 Gy radioactive the mice of the same strain. Then mice were randomly divided into two groups on the next day (day 1) one group (the control group) were injected with S, whereas another group (group SRAW) were injected with TRO-active fraction used in Example 73 (relative activity 44000 in M-e-test) with a dose of 100 μl subcutaneously 1 time per day for 20 consecutive days.

After the start of injection every 6 mice were chosen randomly on the 5th, 10th, 14th and 21st days, blood was taken from their ophthalmic veins and the number of platelets counted in microclimate (type E-2500, Toa Iyo Denshi).

Changes in numbers of platelets are given in Fig.28. So, in all groups the number of platelets decreased over time after injection of bone marrow cells. On the 10th day of the quantities were very low (approximately 3% of the value before applying TDC). After that noted a gradual recovery and on the 4th day platelet counts were 11% and 13% in the control group and in the group SRW, respectively. On the 21st day was celebrated only 37% of the recovery in the control group, whereas in the group of TRO recovery was approximately 65%, and then observed a significant effect of speed recovery. These results confirmed that SRAW man, polucen rapeutique the effect of TRO on thrombocytopenia after irradiation of radioactive rays

36 male mice of strain 1CR eight weeks of age were irradiated by x-rays 5Gy on the whole body and were randomly divided into two groups. The next day (day 1) one of these groups (the control group) were injected with S, whereas another group (group SRAW) were injected with TRO-active fraction used in Example 73 (relative activity 1440000 in M-e-test) once a day subcutaneously for 3 days and the next day the relative activity 360000 (according to the same test) were injected with subcutaneous once daily for 7 consecutive days. After the start of injection, each group was divided into three groups: the group from which blood was taken on the 4th, 11th and 21st days; another group, from which blood was taken on the 7th and 13th days, and the third group, from which blood was taken on the 9th and 15th days. Blood was taken from the ophthalmic veins and the number of platelets counted using microcamera (type F-800, Toa IRE Denshi). Changes in numbers of platelets are given in Fig.29. Thus, in the control group the number of platelets decreased over time after x-ray irradiation and on the 9th day of the quantities were very low (approximately 25% of the value before x-ray irradiation). On the 11th and 13th days of the quantity Tr is elicin before x-ray irradiation. In the group SRW these quantities decreased approximately 24% of the value before x-ray irradiation on the 7th day. After this was pointed out the increase and on the 9th day of the platelet count has recovered to approximately 82%. On the 11th day after the amount was more values before x-ray irradiation and before the 21st day continued this trend. As mentioned above, on the 9th day, when in the control group had seen a decline in the number of platelets in the group SRAW was already changed in the direction of increasing and on the 11th day platelet counts were more values before x-ray irradiation, while remaining high after that. On the other hand, in the control group were required 15 days for recovery of platelet counts to values before x-ray irradiation. These results showed that the TRO person obtained in Example 56, reduces the time required for recovery to normal after the reduction of the number of platelets after x-ray irradiation, and that it exhibits a therapeutic effect on thrombocytopenia after irradiation with x-rays.

Example 78. Action hTO163 to increase platelet count

15 healthy samz the soup of microcamera (type F-800, Toa IRE Denshi), were divided randomly into 4 groups: control group and groups a, b and C. the First group (control group)

were subcutaneously injected with S containing 0.1% serum from mice, 1 time a day for 5 consecutive days. Groups a, b and C were injected with TRO-active fraction of SP Sepharose Fast Flow, obtained in Example 65, with subsequent breeding S containing 0.1% mouse serum, at doses of about 40000000, approximately 8000000 and about 1600000/kg body weight, respectively, according to the relative activity in M-e test NTRO, subcutaneously for 5 consecutive days.

On the 6th, 8th, 10th and 12th days after the injection took the blood from the orbital vein of each mouse and the number of platelets counted using microcamera (type F-800, produced by Toa Iyo Denshi). Changes in quantities of platelets is shown in Fig.30.

Thus, in the control group there was almost no change in the quantities of platelets, whereas in the received TRO groups increase in numbers of platelets were observed in all cases and the highest values were on the 8 th day after injection. Among each of the obtained TRO groups was observed dependence of the response on the dose. So, in the group And increase of about 88% and the rise, then all the results showed a significant difference from the control group (p<0,01 according to Dunnet test). A similar increase was also noted in group b and, although the degree of increase was lower than in group a, the increase of about 65% and about 84% were noted at the 6-th and 8-th day, respectively, with significant difference compared with the control group (p<0,01, Dunnet test). Raising the action in group C was lower than in group C. still observed approximately a 31% increase on the 8th day, when he noted the high number of platelets. These results confirmed that h163 obtained in Example 65, increases platelet counts, and that his action reveals the dependence of the response on the dose.

Example 79. Therapeutic effect hO163 on thrombocytopenia

100 male mice of strain SN/N eight weeks of age were injected with intravenous 50 mg/kg of the hydrochloride of nimustine (ACANU) and then randomly divided into 4 groups: control group and groups a, b and C, each of which consisted of 25 mice. The next day (1st day) control group were subcutaneously injected with 5 ml/kg S once a day for subsequent days, whereas group a, b and C were injected with subcutaneous TRO-active fraction SP S00/kg body weight, accordingly, the values of relative activity h163 in M-e-test once a day for subsequent days.

After the start of injection of mice in each group were divided randomly into 5 groups and blood was taken at the 5th, 8th, 10th, 12th and 14th day, respectively. Blood was taken from orbital vein and the number of platelets counted using microcamera (type E-2500 manufactured by Toa IRE Denshi). Changes in quantities of platelets is shown in Fig.31. Thus, in the control group the number of platelets decreased over time after the introduction of CADI, giving the lowest values (approximately 15-16% of the value before the introduction of ACNU) 8-10 th days, and the degree of recovery was only about 28% and about 51% in 12th and 14th day respectively. On the other hand, in the group And this number decreased to approximately 25% of the values before the introduction of CADI on the 8th day. After that, however, they changed upwards and was restored to approximately 34% to approximately 89% in 10th and 12th days, respectively. On the 14th day the platelet count was greater than before the introduction of CADI.

As mentioned above, the lowest platelet count was observed on day 8 in all groups is my low platelet count. In the control group there was almost no recovery of quantities of platelets even at day 10, whereas in the group treated with TRO person, recovery in platelet count was noted in all these groups, although the levels varied. In the group which was administered 50 mg/kg, recovery was observed on 12th day, when platelet counts were higher than the values before the introduction of ACNU. If you compare this with the fact that in the control group, the recovery was only about 51%, compared with the value before the introduction of CADI, even on the 14th day, it becomes clear that hO163, obtained as described in Example 65, enhances recovery of platelet counts after their reduction. Thus, it was confirmed that hO163 has a therapeutic effect on thrombocytopenia caused by anticancer agents.

The following are examples of pharmaceutical preparations.

Example 80

Fraction TRO person obtained in Example 56, concentrated, subjected to aseptic processing and frozen at -20oWith getting frozen product for use as a drug for injection.

Example 81

Fraction TRO person obtained in Example 56, concentrated, 5 closed with a rubber stopper, getting liofilizirovannoe substance for use as an injectable drug.

Example 82

Fraction TRO person obtained in Example 56, concentrated, subjected to aseptic filtration and placed in a vial of 10 ml, receiving the product for use as an injectable drug.

Example 83

Fraction hO163 obtained in Example 65, concentrated, subjected to aseptic processing and liofilizirovanny at -20oWith getting frozen product for use as a drug for injection.

Example 84

Fraction hO163 obtained in Example 65, concentrated, 5 ml was placed into the vial of 10 ml using aseptic operations, liofilizirovanny at -20oAnd the vial was closed tightly with a rubber stopper, receiving lyophilized product for use as a drug for injection.

Example 85

Fraction hO163 obtained in Example 65, concentrated, subjected to sterile filtration and placed in a vial of 10 ml, receiving the product for use as a drug for injection.

Example 86

Fraction TRO person obtained in Example 57, was concentrated, subjected to aseptic obrabotka.

Example 87

Fraction TRO person obtained in Example 57, concentrated, 5 ml was placed into the vial of 10 ml under sterile conditions, liofilizirovanny at -20oAnd the vial was closed tightly with a rubber stopper, receiving lyophilized product for use as a drug for injection.

Example 88

Fraction TRO person obtained in Example 57, was concentrated, subjected to sterile filtration and placed in a vial of 10 ml, receiving the product for use as a drug for injection.

Example 89. Aplastic plasma of dogs

Heparinization aplastic plasma of the dog ("ARC") or normal plasma dog ("N9") were obtained as described [Mazur, E. and South, K. Exp Hematol. 13: 1164-1172 (1985); Arriaga, M., South K., Cohen. J. L. and Mazur, E. M. Blood 69: 486-492 (1987); Mazur, E., Basilico, D., Newton, J. L., Cohen, J. L. , Charland, C. , Sohl, P. A., and Narendran, A. Blood 76: 1771-1782 (1990)] , except that 450 rad (4,5 j/kg) exposure of the whole body was used for the recipients.

Example 90. Test for the determination of megakaryocytes man

Standard methods for many of the procedures that are described in the following examples, or other suitable procedures are given in the well-known descriptions of the methods of molecular biology such as, for example, Sambrook, et ark (1990).

APK9 and fractionated ARC analyzed for the ability to stimulate the development of megakaryocytes man from precursor cells GD34+, CD34 - selected cells were obtained from peripheral blood cells as described (Hokom, M. N., Choi, E., Nichol, Hornkohl, A., Arakawa, T., and Hunt, P. Molecular Biology of Haematopoiesis 3:15-31, 1994), and incubated in the following culture medium: the medium of Dulbecco modified by the method of Claims (1MDM; G1, Grand Island, NY) supplemented with 1% Glutamine Pen-Strep (Irvine Scientific, Santa Ana, CA) and 10% heparinization, with a low platelet AB human plasma. Also on Wednesday included a 2-mercaptoethanol (10-4 M), pyruvic acid (110 mg/ml), cholesterol (7,8 μg/ml), adenosine, guanine, citizen, uridine, thymidine, 2-detoxication, 2-deoxyadenosine, 2-deoxyguanosine (10 µg/ml each, Sigma); human recombinant insulin (10 μg/ml), human transferrin (300 μg/ml), soy lipids (1%, Boehringer - Mannheim, Indianapolis, 1N); recombinant basic fibroblast growth factor (2 ng/ml), Genzyme, Cambridge, MA); recombinant epidermal growth factor (human) (15 ng/ml) obtained from platelet growth factor (10 ng/ml, Amgen, Inc., Thousand Oaks, CA). CD34-selected cells were seeded at 2105cells/ml of culture medium with a final volume of 15 µl, in the humidified boxes in 5% CO2in the air, recorded directly in the culture wells with 1% glutaraldehyde and incubated with a mixture of monoclonal antibodies (anti-GPIb, anti-G11, (Biodesign) and anti-GP1b (Dako, Carpinteria, CA). Immune response was demonstrated by the detection system with streptavidin-galactosidase (HistoMark, Kirgegaard and Perry). The megakaryocytes, identified by the blue color, considered using investicionnogo microscope at 100X magnification. The results were reported as averages of megakaryocytes per well standard error of the mean (S). In some cases, the data presented in megakaryocytic units/ml, where the degree to which this test has induced the development of megakaryocytes, normalized to positive ARC control for this experiment. One unit is defined as the amount of material, which leads to the same number of megakaryocytes, and 1 μl ARC standards. The activity of thought associated with the MpL ligand, if it were a block of 5-10 µg/ml MPL-X (soluble receptor pl). It was shown that ARC contains the factor (factors) that stimulate the development of human megakaryocytes in this system. CD34-selected cells, incubated with 10% NK9 within 8 days, found a small number OKEI a very large number painted in blue color megakaryocytes.

Fig.32 shows that increasing the concentration of Mpl-X, added to the culture system of human megakaryocytes, increasing image block the development of megakaryocytes. At concentrations l's more than 5 µg/ml the inhibition was complete. In this experiment, CD34-selected cells were stimulated by 5% ARC. This shows that the activity, which interacts with Mpl-X (preferably l ligand), necessary for the development of megakaryocytes person, and implies that the Mpl ligand is present in ARK.

It was further shown that the activity l ligand necessary for the development of human megakaryocytes present in ARK. ARC (135 ml) was diluted 6 times in environment Suits and were applied to the affinity column l Agricultural Unbound material passing through the column fraction) was collected and concentrated to the original volume before analysis. Bound material was suirable in 10 ml of 1 M N1 and 20% of the pool was diafiltrate and concentrated in 4 times before analysis. CD34-selected cells incubated only in an environment that did not develop in the megakaryocytes. Cells incubated in 5% ARK (the same pool, which are applied to the column), developed in 488 megakaryocytes per well. Cells incubated in 10% of the unbound material, new to the hole. As the active load of the column, and the activity lirovannomu pool essentially completely inhibited 5 μg/ml Ml-X in this test.

Example 91. Transfection of murine or human Mpl receptor in the murine cell line A. Murine Mpl receptor

cDNA of murine receptor Ml full length was subcloned in expressing a vector containing a promoter transcription derived from the LTR of the virus Moloney sarcoma of mice. 5 μg of this construct and 1 μg of breeding marker plasmids pWLneo (Stratagene) to-electroporative in 1L-3-dependent murine cell line (32D, clone 23; Greenberger et al., NS 80:2931-2936 (1983)). Cells were cultured for 5 days, were removed and then incubated in selective media containing 800 μg/ml Geneticin (G418, Sigma) and 1 ng/ml murine 1L-3. Surviving cells were then divided into pools 2105cells were cultured to a growing population of cells, which are then analyzed. 6 populations were tested for surface expression of the receptor l using FS analysis using polyclonal rabbit used for preparing antipeptide serum. One population was chosen for FS of sorting (using cell sorting device with excitation fluorescence) using the same used for preparing antipeptide serum. Oznaczenie 35 days the cells were kept at 1 ng/ml murine 1L-3. One of the subclones, A.1, was used for this part of the work.

Century Human receptor l

The cDNA sequence of human MPl receptor full length (Vigon, 1. , et al., NS 89: 5640-5644 (1992)) was subcloned into expressing a vector containing a promoter and a transcription of the virus Maloney sarcoma of mice (the same vector as in the case of mouse receptor). 6 μg of this design and 6 µg anthropically retroviral packaging design (Landau, N. R., Littman, D. R., J. Virology 66: 5110-5113 (1992)) was transfusional in 3106of 293 cells with the use of the kit for transfection Stratagene with Saro4. The same cells again were transfusional after 2 days and again after 4 days. The day after the last transfection of 293 cells were cultured together WITH 1L-3-dependent murine cell line (32D, clone 23; Greenberger et al., PNAS 80: 2931-2936 (1983). After 24 hours 32D cells were isolated and obtained in the form of stripes in the gradient S (ath-o-yte; mills 1nc.). Cells were increased in the amount of 1 ng/ml murine 1L-3 and then selected for growth in the 20% ARK. Cells were subjected to sorting on the expression on their surface receptor using FACS using polyclonal rabbit used for preparing antipeptide serum. These cells were then used in tests.

Example 92. A.1 analysis for definition wide-angle the notches/15 µl total Nr./well) in a-MEM (Gibco) supplemented with 10% fetal calf serum (FCS), Geneticin (800 μg/ml) and 10% pen/strap (Gibco) in serial dilutions 1:1 test samples. After 48 hours was determined under a microscope the number of viable cells per well. One unit of activity was taken a number of activity, which resulted in 200 viable cells per well. The activity of thought associated with l ligand, if it could completely block the addition to the reaction medium 5-10 µg/ml l Agricultural Activity l ligand in ARK was on average 4400539 units/ml aplastic plasma. Unless otherwise noted, the units of activity l ligand was determined in A.1-test.

Tests with cells, transfitsirovannykh genome of human Mpl receptor, conducted in much the same way as with A.1 cells.

Example 93. The demonstration that l-ligand is present in aplastic plasma or serum sources such as mouse, dog, pig and man

MPL-ligand is present in aplastic plasma or sera from mice, dogs, pigs and humans (table 9). Plasma was collected from BDF1 mice with preobladaniem and 12 days post-irradiation (500 rad). Plasma was tested in A.1-test, in which she showed 2000 units/ml activity, which is essentially completely inhibited Mpl-X (10 µg/ml). Irradiated mouse is 3 units/ml Plasma was collected from dogs with preobladaniem and 10 days post-irradiation (450 rad). Plasma was tested as in A.1 test and in tests with the development of human megakaryocytes. Activity was detected and completely inhibited Mpl-X (10 μg/ml) in both texts. Plasma was collected from pigs, predoplachennih 10 days post-irradiation (650 rad). Plasma was tested as in A.1 test and test development of human megakaryocytes. In both tests, she found the activity of Mpl-ligand (inhibiting 10 μg/ml Mpl-X), comparable to the activity found in aplastic plasma of dogs. Were obtained from human serum. This material was collected from patients with bone marrow transplantation. Sera from 6 patients tested in A.1 test, where they showed activity 903 unit/ml, 88% of which was associated with the Mpl-ligand (inhibited 10 μg/ml Mpl-X). Sera from 14 aplastic patients (abnormality) was tested in the test with the development of human megakaryocytes. As a group, they found the main activity, 941 megakaryocytic units/ml, which completely inhibited 10 μg/ml Mpl-X. the data with the mouse 1L-3, to show the specificity A.1 test. Although this recombinant cytokine induces the growth of this cell line, it is not the lock
With the attempt to improve the biological activity, stability and solubility of some derivatives TRO were constructed and expressed in E. coli. These derivatives generated using the amino acid sequence represented in SEQ 1D 11, included: [Met-2,Lys-1, la1, Val3, Arg129] TPO(1-163), in which arginine replaces leucine at amino acid position 129 (also called L129), [Met-2, Lys-1Ala1, Val3, Arg133] TPO(1-163), in which arginine replaces the histidine at amino acid position 133 (also called 133R), [Met-1, Lys-1Ala1, Val3, Arg143] TPO(1-163), in which arginine replaces methionine at amino acid position 143 (also called M143R), [Met-2, Lys-1Ala1, Val3, Leu82] TPO(1-163), in which the leucine replaces the glycine at amino acid position 82 (also called G82L), [Met-2, Lys-1Ala1, Val3, Leu146] TPO(1-163), in which the leucine replaces the glycine at amino acid position 146 (also called G146L), [Met-2, Lys-1Ala1, Val3Pro148] TPO(1-163), in which the Proline replaces series at amino acid position 148 (also called S148), [C-2, Lys-1Ala1, Val3>2
, Lys-1, la1, Val3, Arg115] TPO(1-163), in which arginine replaces glutamic acid at amino acid position 115 (also called Q115R).

Upon receipt of these derivatives TRO as matrix used h6(1-163), described in example 42, and used the following primers synthesized oligonucleotides:

Ll29R: 5'-GG-3' (for AGD-substituted derivative with Leu-129, substituted AGD in the sequence presented in SEQ 1D 11);

H133R: 5'-GGGGGT-3' (for AGD-substituted derivative with is-133, replaced by Arg in the sequence presented in SEQ 1D 11);

M143R: 5'-GGGGGGG-3' (for Arg-substituted derivative with Met-143, substituted AGD in sequence represented by SEQ 1D 11);

G82L: 5'-GGGGGG-3' (for Lu-substituted derivative Gly-82, replaced by Leu in the sequence presented in SEQ 1D 11);

G146L: 5'-ATGCTGGTTCTGGGTTCTACCCT-3' (for Leu-substituted derivative with Gly-146, replaced by Leu in the sequence presented in SQ 1D 11);

S148: 5'-GTTGGGGGGGG-3' (for RHS-substituted derivative with Sr-148, replaced by the RHS in the sequence presented in SEQ 1D AGD in sequence, presented in SQ 1D 11) and

Q115R: 5'-CTGCCGCCACGTGGCCGTACCAC-3' (for Arg-substituted derivative with Gln-115, substituted AGD in the sequence presented in SQ 1D 11).

Mutant plasmids were constructed with the use of the kit for the in vitro mutagenesis Sculptor (Amersham). Fragment b 1 - ind 111 obtained from pCFM536/h6(1-163), described in Example 42, was inserted in the phage vector Bluescript 11SK (-) (Stratagene, California, USA) after splitting Xba 1 and Hind 111 to obtain plasmids S, which is then transformed in E. coli J109. Single-stranded DNA for mutagenesis were obtained from S using helper phage MCO (Takara-Shuzo, Japan). Mutations were introduced into the gene SRW in accordance with the protocols of the supplier using the oligonucleotides listed above. Sequencing was performed with a kit for DNA sequencing (Applied Biosystems, USA) according to the protocols of the supplier and mutation TRO were confirmed.

Fragments b 1 - Hind 111, derived from mutated S, was inserted in the plasmid RAM, split b 1 and Hind 111. Plasmids pCFM536 carrying mutated genes, transformed in E. coli 261 for receiving transformant expressing genes derived TRO.

Cultivation of E. coli 261, transformed mutated F536, Provadiya not less than 1 day. Frozen cell residue (3 g) suspended in 30 ml of 20 mm Tris buffer (pH 8.5) containing 10 mm EDTA, 10 mm DTT and 1 mm PMSF. The suspension was kept on ice and treated with ultrasound for 5 times with an interval of 1 min (at least). Ultrasonic assisted the cells were collected by centrifugation at 15,000 rpm for 10 minutes.

Sediment suspended in 30 ml of 10 mm Tris buffer (pH 8,7) containing 8 M guanidinium, 5 mm EDTA, 1 mm PMSF and restored by adding 50 mg of DTT. After stirring for 1-1,5 h, pH of the solution was brought to 5.0 diluted model HC1 and then cooled the solution to the 4oWith before breeding.

The solution sample was gradually diluted with 1.5 l of 10 mm CAPS buffer (pH of 10.5) containing 30% glycerol, 3 M urea, 3 mm tsistamin and 1 mm L-cysteine, during the night. After stirring the sample for at least 2 days the precipitate was removed by centrifugation at 8000 rpm for 45 minutes. the pH of the solution was brought to 6.8 6M phosphoric acid and the solution was diluted 2 times. The solution was filtered through 2 layers of filter paper 2 (diameter 90 mm, Too filter paper, Japan) and then applied on a column (2,610 cm) of CM-Sepharose Fast Flow (Pharmacia). The column was washed with 400 ml of 10 mm phosphate buffer (pH 6.8) containing 15% glycerol and 1 M urea, and uranov linear gradient from 10 mm phosphate buffer (pH 7,2), containing 15% glycerol to the same buffer containing 0.5 M N1, flow rate 1.0 ml/min, the Elution of protein was observed by absorption at 280 nm and fractions containing the derivative TRO, was confirmed using SDS-PAGE and collected.

After concentration of the fractions TRO (about 30 ml) to 2 ml using a Centriprep 10 (Omicon) mutant TRO was purified further using L with reversed phase (Waters) column Bondasphere, C4 (3,9150 mm). The elution of protein was carried out using a linear gradient of 0.05% TFA in H2About 2 propanol containing 30% of CH3JV and 0.02% TFA, at flow rate 0.5 ml/min for 40 minutes. Under these conditions, derived TRO was loirevalley after approximately 30 minutes.

For biological analysis cleansed thus the sample TRO were dialyzed twice against 1 l of 10 mm phosphate buffer for 2 days. After concentration using a Centriprep 10 (Omicon) approximately 0.1 mg/ml biological activity was evaluated in the M-e test system, as described previously. It was found that all mutants have almost the same activity compared to the activity h6(1-163), standard breakdown TRO (see Fig.33 and 34).

The list of deposits

Escherichia coli (RT-231/DH5):

FERM BP-4564 and STS-M

Escherichia coli (pEF18S-A2/DH5):

004

Mouse-Mouse-hybridoma P55:

FR BP-4563 and STS-S

SNO/1/1/3 / C6:

FERM BP-4988 and STS-S SNOT-63-79-S1:

FERM BP-4989 and STS-S.

1. The polypeptide of thrombopoetin (SRW) having the biological activity of specific stimulus or increasing the formation of platelets containing the amino acid sequence of SEQ ID NO 6, or its derivative, and which may optionally contain amino acids Met-2-Lys-1, Met-1or Gly-1.

2. Polypeptide TRO on p. 1 containing the amino acid sequence 1-332 SEQ ID NO 6.

3. Polypeptide TRO on p. 2 consisting of the amino acid sequence 1-163 SEQ ID NO 6.

4. Polypeptide TRO on p. 2 consisting of the amino acid sequence 1-232 SEQ ID NO 6.

5. Polypeptide TRO on p. 2 consisting of the amino acid sequence 1-151 SEQ ID NO 6.

6. Polypeptide TRO according to any one of paragraphs.2-4, having from 1 to 6 amino acids deleteregvalue.

7. Polypeptide TRO on p. 3, selected from the group consisting of [His33] TPO(1-163), [Arg117]TPO(1-163) and [Gly116]TPO(1-163).

8. Polypeptide TRO on p. 3, selected from the group consisting of [His33, Thi33'Pro34]TPO(1-163), [His33Ala33'Pro34]TPO( Ala116', Arg117] TPO(1-163), [Gly116, Gly116', Arg117]TPO(1-163) and [Ala1, Val3]TPO(1-163).

9. Polypeptide TRO on p. 2, selected from the group consisting of [Thr33, Thr333Ser334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr344Ala345, Gly346, Val347, His348, His349, His350, His351, His352, His353]TPO [Asn25, Lys231, Thr333Ser334, Ile335, Gly336, Tyr337Pro338, Tyr339Asp340, Val341Pro342Asp343, Tyr344Ala345, Gly346, Val347, His348, His349, His350, His351, His352, His353]TPO.

10. Polypeptide TRO on p. 2, selected from the group consisting of [Asn25]TPO [Thr33]SRW.

11. Polypeptide TRO on p. 3, selected from the group consisting of [Ala1, Val3, Arg129] TPO(1-163), [Ala1, Val3, Arg133] TPO(1-163), [Ala1, Val3, Arg143] TPO(1-163), [Ala1, Val3, Leu82] TPO(1-163), [Ala1, Val3, Leu146] TPO(1-163), [Ala1, Val3Pro148]TPO(1-163), [Ala1, Val3, Arg59]TPO(1-163) and [Ala1, Val3, Arg115]TPO(1-163).

12. Polypeptide TRO on p. 3,u146] TPO(1-163), [Pro148]TPO(1-163), [Arg59]TPO(1-163) and [Arg115]TPO(1-163).

13. Polypeptide TRO on p. 2, covalently associated with the polymer.

14. Polypeptide TRO on p. 13, characterized in that the said polymer is a polyethylene glycol.

15. Polypeptide TRO according to any one of paragraphs.1-7, characterized in that it is obtained by the method according to p. 24.

16. Polypeptide TRO according to any one of paragraphs.1-7, characterized in that it is obtained by the method according to p. 25.

17. Polypeptide TRO according to any one of paragraphs.1-7, characterized in that it is obtained by the method according to p. 26.

18. DNA encoding a polypeptide TRO according to any one of paragraphs.1-14.

19. The DNA sequence encoding a polypeptide TRO according to any one of paragraphs. 1-14, having the biological activity of specific stimulus or increasing the formation of platelets selected from the group: a) the DNA sequence shown in SEQ ID NO 196, or complementary to it; (b) DNA sequences encoding the polypeptide according to any one of paragraphs.7-12 and hybridization in stringent conditions with the DNA sequence described in (a), or its fragments.

20. The DNA sequence under item 19, characterized in that it is cDNA.

21. The DNA sequence for p. 19, otlichayetsa synthetic DNA.

23. The DNA sequence encoding a polypeptide TRO, having the biological activity of specific stimulus or increasing the formation of platelets, which includes in the direction of 5' --> 3': (a) DNA encoding glutathione-S-transferase (GST), (b) DNA encoding the peptide recognition by thrombin, and (C) DNA encoding a polypeptide TRO.

24. A method of obtaining a polypeptide TRO on PP.1-14, including the stage of expression of the polypeptide encoded by the DNA under item 18, in a suitable host and the selection of the polypeptide TRO.

25. The method according to p. 24, wherein the polypeptide TRO is a polypeptide under item 1, and the method further includes the elimination Met-2-Lys-1from the selected polypeptide TRO.

26. A method of obtaining a polypeptide TRO, having the biological activity of specific stimulus or increasing the formation of platelets, comprising the stage of expression of the polypeptide encoded by the DNA according to any one of paragraphs. 19-23, in a suitable host and the selection of the polypeptide TRO.

27. A method of obtaining a polypeptide TRO, having the biological activity of specific stimulus or increasing the formation of platelets, comprising the stage of: (a) the expression of the polypeptide encoded by Abadi specified GST (peptide recognition thrombin) polypeptide TRO thrombin; and (d) allocation Gly-1polypeptide TRO.

28. Pharmaceutical composition for treatment-related platelet disorders thrombocytopenia in General and thrombocytopenia induced by chemotherapy, radiation therapy or bone marrow transplantation, comprising an effective amount of the polypeptide according to any one of paragraphs.1-17 in combination with a pharmaceutically acceptable carrier.

29. Treatment-related platelet disorders involving the administration of an effective amount of a pharmaceutical composition for p. 28 to a subject having such disorder.

30. A method of treating thrombocytopenia, introducing an effective amount of a pharmaceutical composition for p. 28 to a subject having thrombocytopenia.

31. A method of treating thrombocytopenia induced by chemotherapy, radiation therapy or bone marrow transplantation in a subject, comprising introducing him an effective amount of a pharmaceutical composition for p. 28.

32. The antibody to the polypeptide TRO obtained by the process comprising the stages of immunization of the animal with the polypeptide TRO according to any one of paragraphs.1-14, and selection of antibody to the polypeptide TRO.

33. The antibody under item 32 to select polypeptid the PP.1-14.

 

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The invention relates to the field of medicine and relates to a composition for inhibition of angiogenesis, monoclonal antibodies, polypeptide and method of inhibiting tumor growth

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the activity IL4

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody

The invention relates to the field of the biotechnology industry, in particular the production of virus-inducer for the production of human leukocyte interferon

The invention relates to biotechnology and Microbiology and is a method of obtaining biomass of recombinant strains of E. coli containing plasmid DNA encoding the biosynthesis of cytokines with the properties of factors, tumor necrosisand(TNF-alpha and TNF-beta), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) human and bearing as a selective marker gene bla (resistance to ampicillin)

The invention relates to biotechnology

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the activity IL4

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to the field of molecular biology, immunology, Microbiology and Virology, and is intended to assess the transcriptional levels of genes activity of interferon (if), IGF-dependent and proliferative cytokines

The invention relates to biotechnology

The invention relates to medicine, refers to a medicinal product in the form of a solution and can be used in ophthalmology, otorinolaryngology, Pediatrics and gynecology

The invention relates to medicine and the pharmaceutical industry and relates to dosage forms containing cytokine
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