Means and method of obtaining hematopoietic protein

 

The invention relates to the field of genetic engineering and can be used in the biomedical industry. By screening the cDNA library of mouse isolated nucleotide sequence encoding a protein(thrombopoietin), which has the ability to stimulate MPL-dependent proliferation or differentiation of precursor cells of the myeloid or lymphoid series. Constructed vectors for the expression of the received coding sequences in yeast and mammals. Culturing the eukaryotic cell hosts, transformed proposed expressing vectors, allows to obtain significant quantities of recombinant thrombopoietin. 8 C. and 5 C.p. f-crystals, 2 ill., 6 table.

This application is a partially continued application No. 08/215,203, filed on March 21, 1994, which is a partial continuation of application No. 08/203, 197, filed February 25, 1994, which is a partial continuation of application No. 08/196,025, filed February 14, 1994. All these applications are pending and are used in this document as a reference.

The background to the invention

Hematopoesis predstavitelnykh cells. This process involves a complex interplay of growth factors complex polypeptides (cytokines), acting through membrane-bound receptors on the desired cells. The action of cytokines leads to proliferation (rapid growth) cells and their differentiation as a response to a particular cytokine, which, as a rule, is respecification and/or stagespecific. The development of a single cell type, such as blood plate (thrombotic), from the receiver cells may require the coordinated actions of many cytokines acting in the desired sequence.

Known cytokines consist of interleukins, such as IL-1, IL-2, IL-3, IL-6, IL-8, and so on; and of the factors stimulating the growth of colonies, such as G-CSF, M-CSF, GM-CSF, erythropoetin (EPO), and so on, As a rule, interleukins act as a vector of immune responses and reactions of ignition. The factors stimulating the growth of colonies, stimulate the proliferation of bone marrow cells, actuate Mature leukocytes and in other ways create a single part of the reaction of the recipient on the control of infection with inflammation and infection and immunological infection.

A variety of cytokines were grown as therapeutic agents. Natechnyh violations. Some of the factors that stimulate the growth of the colonies, was used in combination with chemotherapy for cancer to accelerate the recovery of the immune system of the patient. Interleukin-2,d-interferon and-interferon used in the treatment of some forms of cancer. Actions to stimulate megakaryocytopoiesis and thrombocytopoiesis were found in body fluids thrombocytopenic animals and called in the literature "thrombopoetin (recently discussed by McDonald's in Exp. Hematol. 16:201-205, 1988 and McDonald's in the Am.J.Ped.Hematol.Oncol. 14:8-21, 1992). Despite the research, conducted over three decades, the factor or Factors responsible for these processes have not been clearly characterized, in part due to the lack of good sources, good samples for analyses, and partly due to the lack of information about the place of production. Weak disturbances bleeding (ISI) associated with platelet dysfunction, occur relatively frequently (Beckman, Seminars in Hematology 17:292-305, 1980), as, for example, a number of congenital disorders of platelets, including syndrome Bernard-Solera (deficit Gb), disease of Glanzman (deficiency of GPIIb and GPIIIa), congenital afibrinogenemia (reduced or complete absence of even the number of violations associated with the secretion of platelets, the storage pool deficiency, abnormalities of the way arachidonate trombotsitnoy acid, deficiency of platelet cyclooxygenase and thromboxane synthetase and defects of platelet activation (discussed RAO and Holmstrom, Workshop on Hematology 23:102-118, 1986). To this day the molecular basis of these defects is not clear.

Isolation and study of the properties of proteins of platelets would have given an invaluable opportunity to explain the presence of defects in many dysfunction of platelets. The main obstacle to detailed molecular analysis is the difficulty of obtaining mPHK of platelets or their precursors. Platelets lack nuclei and transcriptions. Mark mPHK associated with platelets, are difficult to isolate, and he often undergoes degradation. Creating libraries to do this required a great amount of platelets, usually from 25 to 250 units of whole blood (Izumi and other OEWG. Natl. Acad.Sci.USA 87:7477-7481, 1989: Wiki, etc., Thrombosis and Haemostasis 61:448-453, 1989; and Wenger and other Blood 73:1498-1503, 1989) or phoresia (short-term use of another organism to move) patients with a high content of platelets in the blood due to thrombocytemia (Roth and others, Biochem.Biophys. Res. Somme. 160:705-710, 1989). When thrombocyto and from the entire spectrum coding platelets.

Another way to create the cDNA library of platelets is to isolate and create a library of mPHK, isolated from megakaryocytes, the immediate precursors of platelets. The megakaryocytes are polyploid cells and may contain mPHK, encoding the entire chromosomal set trombotsitnoy and megakaryocytic proteins. However, it has proved difficult to isolate megakaryocytes, if they are in a fairly large number, and to maintain their relative purity.

Recent advances in molecular biology largely clarified hematopoesis, but at the same time has shown that this process is extremely complex. Although the properties of many cytokines have been identified and many of them have already found clinical use, there remains a need for additional agents that could stimulate proliferation and differentiation of precursor Milovidov and limoilou and production of Mature blood cells. A special need is felt in substances that stimulate the development and proliferation of cells megakaryocytes series, including platelets. There is also a need for substances that could be used in the treatment of various types of cytopenia, including thrombocytopenia, an abnormally low number of violations in the field of platelets. The present invention can satisfy all these needs and provides additional advantages.

A brief statement of the substance of the invention

The present invention is the provision of isolated proteins with hematopoietic activity.

Another objective of this invention is to provide methods of producing proteins with hematopoietic activity, as well as isolated DNA molecules, vectors and cells that can be used in the implementation of these methods.

Another objective of this invention is to provide antibodies that detect the epitope (antigenic determinant) on hematopoietic protein.

Another objective of this invention is to provide methods of stimulating the production of megakaryocytes, platelets and neutrophils in mammals, including humans.

Another objective of this invention is the provision of various tools that can be applied when studying the growth of bone marrow cells, differentiation and proliferation; and when determining the diseases that are characterized by abnormalities of bone marrow cells, their differentiation and proliferation.

According OA) proteins, containing the amino acid sequence of SEQ BD NO:2 from amino acid residue 45 to amino acid residue 196; (C) a protein containing the amino acid sequence of SQ ID NO:2 from amino acid residue 45 to amino acid residue 206; (C) a protein containing the amino acid sequence of SEQ ID NO:19 from amino acid residue 22 to amino acid residue 173; (d) a protein containing the amino acid sequence of SEQ ID NO:19 from amino acid residue 22 to amino acid residue 175; (e) allelic Barinov (a), (b), (C) and (d); (f) homologues of samples (a), (b), (C), (d) or (e), in which the protein stimulates the proliferation and differentiation of precursor Milovidov and lipids. In certain embodiments of the invention, the protein contains the amino acid sequence of SEQ ID NO:2 from amino acid residue 45 to amino acid residue 379 or the amino acid sequence of SEQ ID NO:19 from amino acid residue 22 to amino acid residue 353.

Another feature of this invention is that it provides an isolated molecule polynucleotide encoding the protein, as indicated above. In one embodiment of the invention the molecule polynucleotide is a DNA molecule, the content is or of a nucleotide sequence of SEQ ID NO:18 from nucleotide 64 to nucleotide 519. In other embodiments of the invention this molecule contains a nucleotide 237-1241, 174-1241, 105-1241, 105-722, 174-722 or 237-722 of SEQ ID NO:1 or the corresponding region of SEQ ID NO:18. Further, the invention provides allelic variants of these molecules and DNA molecules encoding the hematopoietic protein molecules which encode a protein that is at least 80% identical in amino acid sequence to a protein encoded by one of these parts of SEQ ID NO:1 or SEQ ID NO:18. There are also molecules complementary to these sequences.

Relative to the other distinguishing feature, the invention provides an isolated DNA molecule selected from the group consisting of (a) insert Tcj RI-Xho I plasmid pZGmpT-1081 (ATCC 69566), (b) allelic variants of the DNA molecules (a) and (C) encoding a protein that is at least 80% identical in amino acid sequence to a protein encoded by (a) and (b), in which an isolated DNA molecule encodes a protein with hematopoietic activity.

Relative to another distinctive characteristic of this invention offers expressroute vector, consisting of the following related action items: stimulator (promoter) transcription; DNA segment, display alnost nucleotides, as shown in SEQ ID NO:1 from nucleotide 237 to nucleotide 692, (b) segments of DNA encoding the hematopoietic protein containing a nucleotide sequence as shown in SEQ ID NO:18 from nucleotide 64 to nucleotide 519; (C) allelic variants of (a) or (b) and (d) segments of DNA encoding the hematopoietic protein that is at least 80% identical to the sequence of amino acids of the protein encoded by (a), (b) or (C); and a transcription terminator.

Relative to another distinctive characteristic of this invention offers a cultured cell into which has been introduced expressing vector, as mentioned above, in which the cell expresses hematopoietic protein encoded by the DNA segment. In certain embodiments of the invention the cell is a fungus (fungal) cell, the cell is mammalian or bacterial cell.

Relative to the other distinguishing feature, the invention proposes the introduction of mammalian cells (not people) in the germ line, which was introduced heterologous segment of DNA that encodes a hematopoietic protein, as described above, in which the cell of the mammal produces hematopoietic protein encoded ukazaniya stimulate the production of platelets in the cell of a mammal. These methods consist in the introduction into the cell of a mammal by therapeutic effective amount of hematopoietic protein selected from the group consisting of (a) a protein containing the amino acid sequence of SEQ ID NO:1 from amino acid residue 45 to amino acid residue 196; (C) a protein consisting of the amino acid sequence of SEQ ID NO:19 from amino acid residue 22 to amino acid residue 173; (C) allelic variants of (a) and (b); and (d) homologues of samples (a), (b) or (C), in which the protein stimulates the proliferation or differentiation of predshestvennikov of Milovidov or limoilou, in combination with a pharmaceutically acceptable Vectra.

These and other distinguishing features of this invention will become apparent after we refer to the following detailed description and the accompanying drawings.

Brief description of drawings

Fig.1 is a partially narrowed map vector pDX. Symbols used SV40 ori, origin of replication of SV40: SV40 E, SV40 amplifier (DNA out of the gene amplifying the expression of this gene); MLP, the main late stimulator (promoter) of the adenovirus; L1-3, tripartite leader sequence; ss, splicing signals; PA, polyadenylation site.-1 cDNA antitripsin; alpha leader sequence of the alpha-factor; metro, the coding sequence of the TRO mouse.

Detailed description of the invention

Before a detailed description of the present invention, it is useful to define the terminology.

Allelic variant: Alternative form of a gene that arise by mutation, or a modified polypeptide encoded by the mutated gene. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides with altered amino acid sequence.

cDNA: Complementary DNA made by reverse transcription of the RNA matrix intermediary or a clone or enlarged copy of such molecules. Complementary DNA may consist of one thread or two threads.

Expressing the vector: a DNA Molecule, linear or circular, which contains the segment encoding the polypeptide, the action associated with additional segments that give him the transcription. Such additional segments include promoter and termination sequences, and can also contain one or more sources of replication, one or more breeding markers, amplifier, signal polyadenylation both. The term "linked" indicates that the segments are arranged such that they can act together, that is, transcription begins in the promoter and proceeds through the coding segment to the terminator.

Gene: a Segment of chromosomal DNA that encodes the polypeptide chain. The gene contains one or more region encoding amino acids, which in some cases are interspersed with non-coding "insertion sequences (introns), together with flanking, non-coding regions, which ensure the transcription of the coding sequence.

Molecules, complementary Molecules of polynucleotides with complementary sequence of bases and with opposite orientation compared to the reference sequence. For example, the sequence 5'ATGCACGGG 3’ complementary to the 5’ CCCGTGCAT 3’.

Promoter: Part of the gene, which begin polimerazoi RNA communication and synthesis mPHK.

As mentioned above, the present invention provides compounds and methods for use in the production of proteins with hematopoietic activity. In this document, the term "hematopoietic" means the ability to stimulate the proliferation and/or differentiation of precursor miloje other J. Cell.Physiol. 116: 198-206, 1983; Metcalfe and others Exp Hematol.15:288-295, 1987. Normally, the bone marrow cells are incubated in the presence of a control sample and test sample. Then these cultures are selected for proliferation and differentiation of cells by visual inspection and/or staining. Especially preferred specimen is MTT calorimetric sample of Mosman (J. Immunol.Meth. 65; 55 to 63, 1983; referred to in this document by reference), described in more detail in the example below.

The present invention is based in part on the detected activity, which stimulates the growth of cells through receptor MPL. This receptor (Souri and other Cell. 73: 1137-1147, 1990) was before this discovery orphan receptor, a natural ligand of which was unknown. Through processes of clone and mutagenesis, described in detail in the examples below, the inventors have grown cell line, which depends on the stimulation path MPL, linked through receptors on the survival and growth and which was capable of autocannon stimulation of the receptor. Was found in the conditioned medium of these independent of interleukin-3 (IL-3) cells, which supports the growth of cells, downregulation of the MPL receptor and the dependent is I IL-3 or IL-4, and that neutralization can occur via soluble type receptor. Then they create cDNA library of the cell line, independent of IL-3. DNA was used for transfection of kidney cells baby hamster (KSS), and transfectants were tested for the ability to stimulate cell proliferation, independent of the MPL. A positive clone was isolated, and was producyrovtsa recombinant MPL ligand. It was found that the recombinant protein stimulates the proliferation of a wide range of precursor Milovidov and limoilou and, in particular, stimulates the production of megakaryocytes and neutrophils from undifferentiated precursor cells in the bone marrow cells. In addition, it was found that the recombinant protein stimulates the production of platelets in the test animals. In light of the above, the protein was named thrombopoetin (SRW).

The present invention provides isolated molecules polynucleotide encoding thrombopoietin. Useful for this molecule polynucleotides contain mRNA, genomic DNA, cDNA, synthetic DNA and DNA molecules generated due to the connection of fragments from different sources. For producing recombinantly TRO preferred mo is W what molecule is removed from its natural genetic environment. Thus, the present invention allows to obtain DNA molecules available from other genes with which they are normally associated. In particular, the molecules are free from peripheral or unwanted coding sequences and presented in a form suitable for use within the generated genetic engineering systems, producing protein.

The sequence of cDNA clones encoding a typical proteins of murine or human TRO shown in SEQ ID NO:1 and SEQ TD NO:18, respectively, and the corresponding amino acid sequence shown in SEQ ID NO:2 and SEQ ID NO:19, respectively. Specialists in this field of science will be clear that the sequence shown in SEQ ID NO:1, 2, 18 and 19 and genomic sequence shown in SEQ ID NO:28 and 29 correspond to a single allele of mouse or human gene, and that assumes the existence of allelic variation. Allelic variations of the DNA sequence shown in SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:28, including those containing silent mutations and those in which mutations lead to changes in the amino acid sequence, fall in the scope of the present invention, as p is m in this area will not be difficult to construct sites who will facilitate and simplify the manipulation of nucleotide sequences using alternative codons.

Mouse and human sequences considered in this application are very important tools for the preparation of isolated molecules polynucleotides encoding TPO proteins from other samples ("samples homologs"). Preferably, such samples homologues contained homologues of mammalian, such as proteins cows, dogs, pigs, sheep, horses, and especially primates. In this area of knowledge is already known how to use the serial information from the first sample to clone the corresponding polynucleotide sequence of the second sample. See, for example, Isabel and other Current Protocols in Molecular Bioligy, John Wiley and Sons, Inc., NY, 1987. DNA molecules of the present invention, encoding TRO, typically 60%, preferably 80%, and can 90-95% or more to be identical to the sequences of SEQ ID NO:1 and SEQ ID NO:18 and their allelic variants.

Thrombopoetin molecules are characterized by the ability to specifically bind to a receptor MPL from the same sample and to stimulate the production of platelets in vivo. In the usual test animal TRO capable unalis distribution of mRNA showed what encoding TRO is present in some human tissues and mouse, and especially his lot in the lungs, liver, heart, skeletal muscle and kidney. Thus, in order to isolate homologues from other samples, a cDNA library, preferably from one of the fabrics, which found properties to produce especially a lot mRNA. In this field of science there are well known ways to create a cDNA library. See, for example, Sambrook and other, Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, 1989 and are there links. In order to determine TRO encoding molecules, this library is being piloted on cDNA of mouse or human, are considered in this application, or its fragment, or on one or more probe DNA on the basis of the considered sequences. Particularly useful are the probes containing oligonucleotide of at least 14 or more nucleotides in length up to 25 or more nucleotides, at least 80% identical to a portion of SEQ ID NO:1, SEQ ID NO:18, SEQ ID NO:28 or complemetary sequences of the same length. It is desirable to probe the library with a few restrictions hybridization, i.e., at about 2SSC (saline and sodium nitrate) and Crocidura detection. Positive clones confirmed by sequence analysis and activity, such as the ability to capture the receptor homologues MPL (i.e., the MPL receptor from the same pattern as cDNA) or to stimulate hematopoiesis of homologue bone marrow cells. The specialist will be obvious that it is possible to use other methods of cloning.

Polynucleotide molecules encoding TSO (including allelic variants and samples homologues of the molecules described in this invention may also be used to isolate a clone of the cell line, which produces the MPL ligand and stimulates the growth of autocrine. In short, irrespective of the cell line, transferservice for the expression of MPL receptor (Wigan and others, Proc.Natl.Acad.Sci. USA 89:5640-5644, 1992; Skoda and others, EMBO J. 12:2645-2653, 1993; and SEQ ID NO:17), then mutagenized and selected factor-independent cells. Then these cells are used as the source of TPO mRNA. The corresponding lines of the factor-independent cells contain the line IL-3-independent BaF3 cells (Palacios and Steinmetz, Cell 41%727-734, 1985; Mathey-Prevost and others, Mol.Cell.Biol.6: 4133-4135, 1986), FDC-P1 (Chapel and other Blood 64:786-790, 1984) and MOI (kiss and other Leukemia 7:235-240, 1993). Cell line-dependent growth factor, can be arranged according to published methods 1980). A typical procedure involves extracting cells from tissues (bone marrow, spleen, liver) and cultivation in normal serum medium, such as RPMI 1640 with 10% bovine serum, 15% horse serum and 10-6M hydrocortisone. With an interval of 1-2 weeks out unattached cells and culture fed with fresh medium. Grown, unattached cells are washed and cultured in a medium with an additional source of growth factors (i.e., RPMI 1640+5-20% WEHI-3 conditioned medium as a source of IL-3). These cells are fed with fresh medium at intervals of 1-2 weeks and rise with the culture. After a few weeks or several months, individual clones are isolated by seeding cells on semi-solid medium (i.e. a medium containing methylcellulose) or by limiting expansion. Factor-dependence of the clones confirmed by cultivation of individual clones in the absence of growth factor. To achieve more frequent cultivation of the factor-dependent cells can be used retroviral infection or chemical mutagenesis. These factor-dependent cells transfections to ekspressirovali receptor MPL, then mutagenized, for example, by chemical treatment the cells are then cultured under conditions in which the survival of a cell depends on the production of growth factor autocrine, i.e. in the absence of exogenous growth factor, which is required for the parent cell. Producirovanie TPO is confirmed by the results of the screening (mass testing), such as checking the air-conditioned environment on the cells, Expressway and inexpressibly the MPL receptor, or by checking the activity of conditioned medium in the presence of soluble MPL receptor, or antibodies, in contrast to the known cytokines.

The present invention also provides an isolated protein which is essentially homologous to the protein of SEQ ID NO:2 or SEQ ID NO:19 and the sample sequences. By "isolated" refers to the protein which is in terms other than its natural environment, from the blood or animal tissue. Preferably, the isolated protein was essentially free from other proteins, particularly other proteins of animal origin. It is desirable to obtain proteins of high purity, i.e., more than 95% purity, and even more preferably with a purity level of more than 99%. The term "essentially homologous" is used here to indicate that proteins with 50% consequently the th sequence, shown in SEQ ID NO:2 or SEQ ID NO:19 or samples of their homologues. Consistent identity, expressed in percent, is determined by known methods. SEE, for example, Altshul and other Bull. Math. Bio.48:603-616, 1986 and Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimizatsii alignment using the account, as in sports, the period of 10 corresponds to the projection 1 and the matrix of Jenickova and Jenickova "flowering 62, as shown in Table 1 (amino acids are indicated by standard one-letter codes).

Then is calculated as follows identity, expressed as a percentage:

In fact homologous proteins are characterized by the fact that they have one or more substitutions, delici or additions of amino acids. Such changes it is desirable to have a minor (side) in nature, that is, to be conservative substitutions of amino acids that does not greatly affect the laying or activity of the protein (see Table 2); with small deliciae, usually from one to about 30 amino acids; and small length amino or carboxyl terminal, as, for example, a methionine residue amino-terminal, small creolisation path (space) antigenic epitope or binding domain. Cm. mostly Ford and other Protein Expression and Purification 2:95-107, 1991, which is used in this application by reference.

Amino acids in TRO can be detected using methods known in this field of science, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and wells, Science 244, 1081-1085, 1989). In the latter method one-alanina mutation is introduced at every residue in the molecule, and the resulting molecules mutants tested on Biologicheskie activity (e.g., receptor binding, in vitra or in vivo proliferative activity to identify amino acid residues that are important for activity of the molecules. Sites of interaction between ligand and receptor can also be determined by analysis of crystal structure, which is determined by methods such as nuclear magnetic resonance, crystallography or photoaffinity tagging. See, for example, In de and other Science 255:306-312, 1992; Smith and others, J. Mol.Biol. 224:899-904, 1992; Wlodawer and other FEBS Lett. 309:59-64, 1992.

Usually predict the cytokines 4-alpha-helical structure in which the first and fourth spiral are the most important in the interaction of ligand and receptor and of all members of the family are best preserved. Looking the cytokine sequences suggests, these spiral bound amino acid residues from 29 to 53, 80 and 99, 108 and 130 and 144 and 168, respectively (boundaries are +4 residue). Border spirals mouse (SEQ ID NO:2) and other inhuman TRO can be determined by alignment with the human sequence. Another important structural feature of the TRO is that SRW contains cysteine residues at the points 51, 73, 129 and 195 of SEQ ID NO:2 (in accordance with point 28, 50, 172 106 and SEQ ID NO:19).

In addition, the proteins of the present invention (or a polypeptide fragments) can be combined with other bioactive molecules, especially with other cytokines, for the creation of multifunctional molecules. For example, the region C-terminal thrombopoetin can be connected with other cytokines to enhance their biological properties or improve production efficiency. Molecule thrombopoietin was composed of two areas. The first (amino-terminal) domain of approximately 150 amino acids similar in size to erythropoietin and some other hematopoietic cytokines and like them in the structure. This first area is the second of about 180 amino acids, the structure of which is not very similar to any known in the database structure about the a and threonine, which are the mark glycosylation sites with O-links. This is obviously a high content of carbohydrate suggests that this area should participate in the creation of more soluble the first hydrophobic region. Experiments indicate that the carbohydrate associated with the second area, is in need of intercellular ordered structure and secretion of the protein during biosynthesis. The second area may also participate in the stabilization of the first area against proteolytic degradation and/or extend the half life of the molecule and can make possible the transfer of biological signal or the specific activity of the protein.

Thus, the present invention proposes a series of new, hybrid molecules in which the second area SRW connected by a second cytokine. Preferably the connection region C-terminal SRW with the end of the second cytokine. Preferably the connection be done by splicing at the DNA level in order to allow the expression of hemeroby molecules in systems recombinant production. The resulting molecules are then analyzed to check properties such as increased solubility, increased stability, longer half life time or podat those in which the second area SRW connected with the end of the EPO, G-CSF, GM-CSF, IL-6, IL-3, IL-11. As mentioned above, this is usually done by merging the DNA. Fused cDNA then zablokiruete in the appropriate expressing vector and transformed or transfections in cell owners or organisms in the usual ways. Obtained after the fusion proteins are purified normal chromatographic purification methods (e.g., a chromatography method), and their properties are compared with properties of its own, Nelidovo parental cytokine. Further, such a hybrid molecule may contain additional amino acid residues (e.g., polypeptide linkers) between the components of proteins or polypeptides.

In addition to hematopoietic proteins discussed above, the present invention relates to fragments of these proteins and isolated polynucleotide molecules encoding these fragments. Of particular interest are the fragments of a length of at least 10 amino acids that are associated with the receptor MPL, and polyureathane molecule, at least 30 nucleotides that encode such polypeptides. The polypeptides of this type are detected by the known methods of screening, such as the digestion of intact and this last one) optionally in combination with the methods of structural analysis discussed above. The resulting polypeptides are then tested for the ability to specifically bind the MPL receptor and stimulate the proliferation of cells via receptor MPL. The presence of bundles is determined by known methods, such as those described by Klotz, Science 217:1247, 1982 ("Scatchard analysis"). In short, radiolucency the test polypeptide is incubated (cultivated) with MPL cells bearing the receptor, increasing the concentration of its TRO. Tagged with boundary cells, the polypeptide is separated from negrinho labeled polypeptide by centrifugation through ftalanowe oil. The relationship of the test polypeptide is determined by drawing a graph of the dependence of boundary-free labels (y-axis) from the boundary labels (x-axis). The specificity of the Association is determined in comparison with cytokine other than TPO. Binding of receptors can also be determined by precipitation of the test composition with the help of devoid of movement of the receptor MPL (or associated with ligand extracellular region). In short, the receptor or protein deprived of mobility on the insoluble support. The test composition is marked, for example, metabolism tagging (for example, radio-iodination). Then labeled composition is combined with devoid of motility receptor, unbound substance is removed and is found bound, labeled compound. In this field of science known methods of detecting a variety of labels. Stimulation of cell proliferation is determined in the usual way by calorimetric MTT analysis of cells with receptor MPL. Polypeptides are analyzed for activity at various concentrations, usually in the range from 1 nm to 1 mm.

There are also larger polypeptides up to 50 or more residues, preferably 100 or more residues, more preferably 140 or more residues, the size of the Mature protein. For example, the results of the analysis and modeling of the amino acid sequence shown in SEQ ID NO:2 from residue 51 to residue 195, inclusive, or in SEQ ID NO:19 from residue 28 to residue 172, inclusive, suggest that these parts of the molecules are areas such as cytokine, capable of samoorganizovatjsya. Represent the interest of a molecule containing this basic cytokine-like area, plus one or more additional segments or areas of the product of the primary broadcast. Thus, others are interested in what the experts in this field of science is obvious, that intermediate forms of the molecules (for example, those that have With-the ends between residues 196 and 206 in SEQ ID NO:2, or those that have N-ends between residues 22 and 28 in SEQ ID NO:19) are of interest, as well as polypeptides with one or more amino acid substitutions, deletions, insertions, or N - or C-terminal length described above. Thus, the present invention provides hematopoietic polypeptides with at least 10 amino acid residues, preferably at least 50 residues, more preferably at least 100 residues, and particularly preferably, at least about 140 residues in length, in which these polypeptides are actually homologous relatively the same size of the polypeptides of SEQ ID NO:2 or SEQ ID NO:19.

The proteins according to this invention can be produced in the cells of the host, created by genetic engineering, by known methods. Appropriate cell hosts include cells of this type, which can be transformed with exogenous DNA and grown in a culture containing bacteria, fungal cells and cultivated in higher eukarioticalkie cells. Technology manipulation of cloned molecules On the r Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel, etc. that are used in this application as a reference material.

Typically, the DNA sequence encoding the protein according to this invention, the action associated with the promoter transcription and terminator within expressroute vector. This vector should usually contain one or more electrovanne markers and one or more started replication, although expert it is clear that within certain systems electrovanne markers can be created on separate vectors and replication of exogenous DNA can be performed by integrating into the genome of the host cell. Selection of promoters, terminators, selective markers, vectors and other elements is a matter of the usual calculation for mid-level professionals. Many of these items are described in the literature and can be removed through commercial vendors.

In order to direct the protein according to this invention on the secretory path of the host cell, expressing the vector of the expected sequence of secretory signals (also known as a leader sequence or pre sequence). The sequence of the secretory signal connects to the DNA sequence, Kozlov is usually located at an angle 5’ to the DNA sequence, encoding the desired protein, although a certain sequence can be located anywhere desired DNA sequences (see, e.g., Welch and others, U.S. Patent No. 5,037,743; Holland and others, U.S. Patent No. 5,143,830). The sequence of secretory signals may be from those normally associated with the protein according to this invention, or may be from a gene encoding another selected protein.

Yeast cells, particularly cells of the genus Sacharomyces, are the preferred cells of the host used in this invention. Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins of them considered, for example, Kawasaki in U.S. Patent No. 4,599,311; Kawasaki and others, U.S. Patent No. 4,931,373; Break, U.S. Patent No. 4,870,008; Welch and others, U.S. Patent No. 5,037,743 and Murray and others, U.S. Patent No. 4,845,075 that are included in this document as reference material. Transformed cells are selected on phenotype determined by selektirovaniya the marker for resistance to drugs or ability to grow in the absence of specific nutrients (e.g., leucine). For use in yeast is the preferred vector is a vector TCI growth in glucose-containing medium. The preferred sequence of secretory signals for use in yeast is the sequence of S. cerevisiae MFal gene (Break ibid; Kurgan and other U.S. Patent No. 4,546,082). Suitable promoters and terminators for yeast are these elements of genes glucokinase enzyme (enzyme) (See, for example, Kawasaki, U.S. Patent No. 4,599,311; Kingsmen and other U.S. Patent No. 4,615,974; and Bitters, U.S. Patent No. 4,977,092, which are included in this document as a reference material) genes and alcohol dehydrogenase. Cm. also U.S. Patent No. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which is also incorporated herein as reference material. Conversion systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces mb, Ustilago maydis, Pichia pastoris, Pichia guillermondi and Candida maltosa, known in this area. See, for example, the work Glisson, etc., J. Gen.Environ. 132:3459-3465, 1986 and Cregg, U.S. patent No. 4,882,279.

Other fungal cells are also suitable as host cells. For example, cells Aspergilus can be used according to the method of McKnight and others, U.S. Patent No. 4,935,349, which is used in this document as reference material. How to convert Acremonium chrysogenum discusses Sumino and others, U.S. Patent No. 5162,228, which is used in this Doucette reference material.

Preferred cells are hosts under this invention are cultured mammalian cells. Methods of introducing exogenous DNA into the cells of the host mammal include transfection by calcium phosphate (Wingler etc., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and van der EB, Virology 52: 456, 1973), electroporation (Newman and others, EMBO.7.1:841-845, 1982) and the transfection by DEAE-dextran (Ausubel and other Current Protocols in Molecular Biology, John Widly and sons, Inc, NY, 1987), which is incorporated herein as reference material. The production of recombinant proteins in cultured mammalian cells examined, for example, Levinson and others, U.S. Patent No. 4,713,339; Hagen and others, U.S. Patent No. 4,784,950; Palmiter and other U.S. Patent No. 4,579,821 and Rinholda, U.S. Patent No. 4,656,134, which are incorporated herein as reference material. In cultured mammalian cells include COS-1 (ATCC No CRL 1650), COS-7 (ATSS No CRL 1651), BHK (ATCC No CRL 1632), BHK 570 (ATCC No CRL 10314), 293 (ATCC No CRL 1573; Graham and others, J. GEn.Virol. 36:59-72, 1977) and cell lines of Chinese hamster ovary (e.g., Cho-K1; ATSS No CCL 61). Known and other suitable cell line, which can get in public repositories, such as Amerikanski, such promoters as of SV-40 or cytomegalovirus. Cm. for example. U.S. patent No. 4,956,288. Other suitable promoters are composed of promoters from metallothionein genes (U.S. Patent No. 4,579,821 and 4,601,978, which are included in this document as a reference material), and late major adenoviral promoters.

Selection with the help of drugs commonly used in the selection of cultured mammalian cells in which the introduced foreign DNA. These cells commonly referred transfectants". Cells that are cultured in the presence of selective substances and is able to transfer the desired gene to its offspring are called "Stable transfectants". Preferred selective marker is a gene encoding resistance to neomycin antibiotic.

The selection is carried out in the presence of a drug type neomycin, such as G-418 or the like. The selection system may also be used to enhance the expressiveness of the desired gene, a process called "amplification". Amplification is carried out by cultivation of transfectants in the presence of a small amount of selective substances with subsequent increase in the number of selective agents for selection khatoum marker is reductase ligitimate, which gives resistance to methotrexate. Other genes resistant to therapeutic drugs (e.g., resistance to the action of hygromycin, resistance to many medicinal drugs or promicious acetyltransferase), can also be used.

Other eukaryotic cells can also be used as cell hosts, including insect cells, plant cells and cells of the birds. Transformation of insect cells and production of these alien proteins described in the work of Guarino and other U.S. Patent No. 5,162,222; Bang and other U.S. Patent No. 4,775,624; and in WIPO publications WO 94/06463, which are used here for reference. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells have been described in the Sinker and others J. Biosci. (Bangalore) 11:47-58, 1987.

Preferred prokaryotic cell hosts for use in this invention are strains of bacteria Escherechia coli, although it is also applicable Bacillus and other genera. Conversion technology these host cells and expression of sequences of foreign DNA cloned from them, are well known (see, for example, the work of Sambuca etc. ibid.). When expression of the protein in bacteria, such as E. Li, protein can maintain the remedy bacterial secretion sequence. In the previous case, the cells lyse and the granules are recovered and denature by using, for example, guanyinge isothiocyanate. The denatured protein is then formed by dilution denaturant. In the latter case, the protein can be recovered from periplasmic region in a soluble and functional form by breaking cells (e.g., by sonication or osmotic shock) in order to release the contents of periplasmatic area and to recover the protein.

Converted or transfection cells cultivated hosts in accordance with known methods in cultivating an environment consisting of nutrients and other components required for growth of the selected host cells. In this field of science known to a large number of suitable environments, including environments of a particular composition and complex environment, which usually contain a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The medium may also contain components such as growth factors or serum, as needed. The growth medium is typically selected for cells containing exogenously added DNA, for example, the choice of therapeutic drug or neare, or transfections cells of the host.

In the framework of the present invention for producing TRO is possible to use transgenic technology. It is preferable to produce the proteins in the mammary glands in the female recipient mammal. Expression in the mammary gland and subsequent secretion of the desired protein in the milk helps to overcome many of the difficulties encountered in the isolation of protein from other sources. Milk is easy going, can be in large quantities and has a good biochemical characteristics. In addition, the milk, there is a high concentration of major milk proteins (from about 1 to 15 g/l).

From a commercial point of view it is obvious it is preferable to use as the recipient of the sample with a large volume of milk production. Although you can use small animals like mice or rats (and they preferred at the stage of proof of concept), in the framework of this invention it is preferable to use livestock, including pigs, goats, sheep and cattle, not limited to the listed animals. Especially preferred are the sheep, by factors such as previous history of transgenesis is oloka. Cm. WI publication WO 88/00239 for comparison of factors affecting the choice of samples recipients. Usually it is desirable to select such animals-recipients that were reared as dairy breeds, such as the East Fridlandskiye sheep, or to enter the dairy breeds of cattle breeding of transgenic lines at a later time. In any case, it is necessary to use animals in good health.

In order to get the expression in the mammary gland, is used transcriptional promoter of the gene of milk protein. Genes milk protein contain genes encoding casein (see U.S. Patent No. 5,304,489 included in this document as ssylochku material), beta-lactoglobulin, alpha-lactalbumin and acidic protein. Here is preferred promoter of beta-lactoglobulin (BLG). In the case of gene Ovine beta-lactoglobulin, you can usually use a portion of the flanking sequence of the gene, at least, 406 bp 5’, although a more preferred are a larger part of the 5’ flanking sequences up to 5 kpb, say, -4,25 kpb segment of DNA that surrounds the 5’ flanking promoter and non-coding part of the gene of beta-lactoglobulin. Cm. work Whitelaw and others, Biochem J. 286:31-Asti gene beta-lactoglobulin, as the genomic region of the gene to be expressed. Usually in this field of science is accepted that design, devoid of introns, for example, Express a bad compared to constructs containing the DNA sequence (see Brinster and others, Proc.Natl.Acad. Sci. USA 85:836-840, 1988; Palmer and others, Proc.Natl.Acad.Sci.USA 88:478-482, 1991: Watlow and other Transgenic Res.1:3-13, 1991; WO 89/01343: WO 01/02318). In light of the above, usually it is preferable, where possible, to use genomic sequence containing all or some of their own introns of the gene encoding the desired protein or polypeptide, Thus, it is preferable to further the introduction of at least some nitroav, for example, preferred is a gene of beta-lactoglobulin. One such area of a segment of DNA that provides the splicing of Nitron and polyadenylation, RNA from the 3’ non-coding region of the gene of beta-lactoglobulin sheep. When replacing natural 3’ non-coding sequences of a gene that beta lactoglobulin segment sheep can both promote and stabilize the level of expressiveness of the desired protein or polypeptide. In other embodiments of the invention, the region surrounding the start ATG sequence TRO, semesterly for imaginary initiate tissue to enhance expressiveness. Usually replace the whole shall sequences TRO and 5’ non-coding sequence, for example, the BLG gene, although you can replace and smaller area.

For the expression of TPO in transgenic animals segment of DNA that encodes a TRO, connected in deystviye with additional DNA segments required for the expression, in order to produce a unit of expression. Such additional segments contain the above-mentioned promoter and sequences, which create conditions for the termination of transcription and for polyadenylation mPHK. Further, these units expression must contain a segment of the DNA sequence encoding the secretory signal in action coupled with segment coding for TRO. The sequence of the secretion signal may be a native sequence TRO or may be a sequence of secretory signals of another protein, such as, for example, the protein of milk. See, for example, the work of von Genga, Nuc.Acids ReS& 14% 4683-4690, 1986; and Ministry of Foreign Affairs and other United States Patent No. 4,873,316, which are incorporated herein as reference material.

Building units of expression for use in transgenic animals is usually performed by inserting a sequence TRO in plasmida actually using any sequence of ligatures. In particular, the known method is the use of a vector containing a DNA segment encoding a milk protein, and replace the coding sequence for milk protein on the coding sequence of TPO polypeptide, thereby creating a gene fusion, which includes sequences regulating gene expression of milk protein. In another case, the cloning units expression plasmid or other vectors simplifies the amplification sequence TRO. Amplification is typically performed in bacterial cells-hosts (for example, E. Li), thus, these vectors usually contain the beginning of replication and breeding token acting in bacterial cells-hosts.

Then the unit of expression is introduced into a fertilized egg (including embryos at an early stage) of the sample selected recipient. Introduction heterologous DNA can be done in several ways, including microinjection method (see U.S. Patent No. 4,8736191) retroviral infection (genes, Science 240:1468-1474 being more accurate, 1988) or by site-directed integration using primary cells of embryo stem (ES) (discussed in the work of Bradley and other Bio/Technology 10:534-539, 1992). Then these eggs are implanted in aizanoi in the germ line, can transfer DNA to their offspring usual way of Mendel, which gives the opportunity to develop transgenic herd.

In this field of science known main methods of producing transgenic animals. See, for example, the work of Hogan and others, Manipulating the Mouse Embryo: A laboratoty mnul, Cold Spring Harbor Laboratory, 1986; work Simonov and others BIol.REprod.32: 645-651, 1985; the work of Buhler and other Bio/Technology 8:140-143, 1990; the work of Ebert and other Bio/Technology 9:835-838, 1991; the work of Krimpenfort and other Bio/Technology 9:844-847, 1991; the wall and other J. Cell.Bilochem. 49:113-120, 1992; U.S. Patent No. 4,873,191 and 4,873,316; WIPO Publication WO 88/00239, WO 90/05188, WO 92/11757; GB 87/00458, which are incorporated herein as reference material. Methods of introducing foreign DNA sequences in mammals and in their germ cells were first tested on mice. See, for example, the work of Gordon et al. in Proc.Natl.Sci.USA 77:7380-7384, 1980; the work of Gordon and Ralla. Science 214% 1244-1246, 1981; the work of Palmiter and Brinster, Cell 41:343-345, 1985; the work of Brinster etc. Proc.Natl.Acad.Sci.USA 82:4438-4442, 1985; and the work of Hogan and others (ibid.). These methods have been adapted for use in a large number of animals, including cattle (see, for example, WIPO Publication WO88/00239, WO 90/05188 and WO 92/11757; and the work of Simon and others, Bio/Technology 6:179-183, 1988). In conclusion, it is scasato cattle is the introduction of several hundred linear molecules of the desired DNA into one of the cores of a fertilized egg in the standard in this field of science. We also used the injection of DNA into the cytoplasm of the zygote.

You can also use the production in transgenic plants. It is possible to generalize or to direct the expression of a specific organ such as the tuber. Cm. the work of Hiatt, Nature 344:469-479, 1990; the work of Edelbaum and other J. Interferon Res.12: 449-453, 1992; the work of Simons and other Bio/Technology 8:217-221, 1990; and the publication of the European Patent office EP 255,378.

The solid prepared according to this invention, purified by the known methods, for example, effendim purification and separation by size, charge, solubility and other properties of the protein. When the protein enduringly in cultured mammalian cells, it is desirable to cultivate these cells in a medium free from serum in order to limit the amount of impurity (contaminant) protein. The preferred method of fractionation is affinity chromatography on concanavalin And or other pectin, thus using the carbohydrate present on the protein. These proteins can also be purified using motion-impaired MPL receptor protein or portion thereof, associated with the ligand, or by using affinity tag (e.g., polyhistidine, substance P, or another polypeptide or protein for which there is and is ecially site crushing.

The proteins according to this invention can be used therapeutically wherever you need to increase cell proliferation in the bone marrow, for example, in the treatment of cytopenia, which caused alifaticescoe anemia, myelodysplastic syndromes, chemotherapy or congenital cytopenia; in patients with bone marrow transplantation; patients with transplantation of peripheral blood stem cells; and in the treatment of conditions that cause failures in the functioning of the bone marrow, such as myelodysplastic syndrome. Proteins are also suitable for increasing the production of platelets, for example in the treatment of thrombocytopenia. Thrombocytopenia is associated with a large group of diseases and clinical situations, which can independently or in combination to create such conditions. Indications of low platelet count can be the result of, for example, defects in the production of platelets (due to, say, congenital disorders, such as Fanconi syndrome, syndrome of absence ..., anomaly Viskota Aldrich, the anomaly may Heklina, syndromes Bernard-Soulie syndrome Minneapolis syndrome Epstein, trombotsitnoy syndrome Montreal and syndrome Eckstein), abnormal distribution thrombocyto the formal isolation of platelets in the spleens of patients with an enlarged spleen (e.g., because of ciroza or overexertion of the heart). For example, the dosage of the chemotherapy drugs used in cancer therapy, can suppress the development of progenitor cells platelets in the bone marrow, resulting in emergent thrombocytopenia limits chemotherapy and might strongly require a blood transfusion. In addition, to ease the production and distribution of platelets can some malignancy. Radiation therapy is designed to kill cancer cells, killing, and also cell precursors of platelets. Thrombocytopenia may also occur because of various autoimmune disorders in platelets, caused by the action of therapeutic drugs, alloimmunity newborn, alloimmunity transfusion of platelets and viral infection (including human virus T cells). The proteins according to this invention can reduce or eliminate the need for transfusion, making it possible to reduce the probability of alloimmunity platelets. Abnormal disorders of platelets can occur because: (1) increased consumption of platelets by transplantation vascular tissue and traumatized tissue, (2) immune mechanism, associated with, n is Otopeni (ITP), autoimmune diseases, hematologic disorders such as leukemia or lymphoma or metastasis cancer in the bone marrow. Other indications for the use of the protein according to this invention is elasticise anemia and suppression of the bone marrow, caused by the action of therapeutic drugs, for example, by chemotherapy or treatment of infection by the human virus T-cell AZT.

Thrombocytopenia is manifested in increased bleeding, such as bleeding of the mucous tissue of the region of the nasopharynx or the gastrointestinal tract, as well as secretions from wounds, ulcers and places of injection.

When pharmacological use of proteins according to this invention are recommended for parenteral injection, in particular intravenously or subcutaneously already known methods. Intravenous administration should be carried out by injection or by infusion over a period of from one to several hours. Usually pharmacological purposes include hematopoietic protein in combination with a pharmaceutically acceptable vector, such as saline solution, saline solution containing buffer, 5% dextrose in water, etc. In the next assignment can be one or more of excipients, conservance, hematopoietic proteins according to this invention can be combined with other cytokines, especially early-acting cytokines, such as factor receiver cells, IL-3, IL-6, IL-13 or GM-CSF. When using such a combined therapy cytokines can be combined in one structure or can be entered in a separate structure. How to create structures well known in the field of science and disclosed, for example, in the work of Gennaro and other Reminoton''s Pharmaceutical Science, Mack Publishing Co., Easton, PA, 1990, which is incorporated herein as reference material. Therapeutic doses usually range from 0.1 to 100 mg/kg of the weight of the patient every day, preferably from 0.5 to 20 mg/kg / day, the dose should be accurately determined by the physician according to accepted standards, taking into account the nature and complexity of the conditions that must be treated, characteristics of the patient and so on dose Determination is based on the average qualification level of a specialist in this field. Proteins usually need to be entered during the period up to 28 days after chemotherapy or bone marrow transplantation or until until the platelet count reaches >20,000/mm3preferably >50,000/mm3. Usually proteins waktunya the amount of waste is the quantity sufficient to produce clinically significant increases in proliferation and/or differentiation of precursor cells of limoilou or Milovidov, which will be manifested in the increase in circulating levels of Mature cells (e.g. platelets or neutrophils). Thus, treatment trombotsitnoy violation shall continue until such time as the measure of the number of platelets will not come, at least up to 20,000/mm3preferably up to 50,000/mm3. The proteins according to this invention can also be administered in combination with other cytokines, such as IL-3, -6 and -11; factor receiver cells; erythropoietin; G-csf and GM-csf. In the framework of modes of combination therapy with a daily dose of other cytokines, as a rule, should be: RPO, <150 U/kg; GM-CSF, 5-15 mg/kg; IL-3, 1-5 mg/kg, and G-csf, 1-25 mg/kg, for Example, combination therapy with EPO is indicated for anemic patients with low levels of EPO.

Proteins in this izobreteniya are also a valuable tool to study in vivo the differentiation and development of hematopoietic cells, for example to explain the mechanism of cell differentiation and determination of the sequence of cellular generations of cells of Mature cells, and may also find application as proliferation, the example at autologous cultivation of bone marrow. In short, the bone marrow is taken from the patient before chemotherapy and processed TRO, optionally in combination with one or more other cytokines. The treated bone marrow is then returned to the patient after chemotherapy to accelerate recovery of the bone marrow. In addition, the proteins according to this invention can also be used for in vivo increase in the volume of bone marrow or peripheral blood progenitor cells (RVRS). Before chemotherapy, the bone marrow can be stimulated by a factor of barrel cells or G-csf for release early progenitor cells in the peripheral circulation. These precursors can be collected from peripheral blood and concentrate, and then treated in culture with TPO, optionally in combination with one or more other cytokines, including SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11, for differentiation and proliferation in megakaryocyte culture with high density, which can then be returned to the patient after holding high-dose chemotherapy.

There are also antibodies that are associated with the epitope on the protein according to this invention. Such antibodies can be produced raznoobraznogo known and can be performed, for example, immunotherapies animal highly purified protein or polypeptide fragment. It is also desirable to introduce a protein or polypeptide in combination with adjuvant (stimulator), such as adjuvant Fronde, in order to strengthen the immune response. Although a single injection of antigen may be enough to cause producirovanie antibodies in an animal, usually preferably the injection of a large dose, followed by one or more repeated injections over a period of time from several weeks to several months. Cm. the work of Harel and other Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Roca Raton, FL, 1982, which is incorporated herein as reference material. Then blood is taken from the animal and coagulums (coils), and the antibodies isolated from the serum using a known method, for example, precipitation of salts, chromatography with ion exchange, affinity chromatography or high performance liquid chromatography.

Usually it is preferable to use monoclone antibodies compared with polecanymi the antisera. Monoclone antibodies have the advantage of simpler production, specificity, and reproduction. Ways of Diller and Milstein (Nature 256:495, 1975 and in Eur.J.Immunol. 6:531-519, 1976). Cm. also the work of Jarella there and HART U.S. patent No. 5,094,941, which are incorporated herein as reference material. In short, cells producing antibodies obtained from immunized animals, immortalized and otrivautsa, or first protsezhivayut for producing antibodies, which is associated with the TRO. Positive cells then immortalized merge with the cells of the spinal cord. Non-human antibodies can be "humanized" by the known methods. See, for example, U.S. Patent No. 4,816,397: the publication of the European Patent Office 173,494 and 239,400 and WIPO WO 87/02671 and WO 90/00616, which are incorporated herein as reference material. In short, permanent regional human genes are connected with the corresponding inhuman variables regional genes. For example, amino acid sequences that represent the binding sites of the antigen (CDR or complementary-determining regions) of the parent (inhuman) monoclonal antibody are grafted on DNA level on human variables regional frame of the sequence. Methods for doing this are known and considered, n is 6: 10029-10033, 1989). Then the combined genes transfections in cell-hosts, which are grown in accordance with known methods. In another embodiment, monoclonal antibody producing cells can transfectants with cloned human permanent regional genes and genes Kemerovo antibodies generated by homologous recombination. Thus, it is possible to construct monoclonal antibodies, a significant portion of which will be human, thereby creating antibodies that are more suitable for multiple injection patients-people.

Antibodies with single chain can be grown by the expression of the recombinant polypeptide, which usually consists of a sequence variable light chain that is connected usually via a linker polypeptide - variable sequence of the heavy chain. In this field of science known methods of predatirovaniya antibodies, single chain and are disclosed, for example, in the work of Davis and others (Bio/Technology 9:165 to 169, 1991).

Antibodies that are associated with epitope TRO, useful, for example, when diagnosing diseases characterized by low platelets, megakaryocytes or other blood cells or their predshestvenniki diagnosis, usually placed at the base of the results of the study of blood and plasma using known methods of immunological analysis, such as immunoabsorption analysis of related enzymes or radioimmunoassay analysis. Analyses of this kind are well known in this field of science. See, for example, the work of HART and others, Biochem. 29:166-172, 1990; Ma and others, British Journal of Haematology 80:431-436, 1992; and the work of Andre and others Clin.Chem. 38/5:758-763, 1992. Diagnostic analysis of the activity of TPO can be useful in determining the patient population most likely being more convenient than SRW therapy. Antibodies to TPO can also be used for purification of the TRO, for example for fixing antibody on a solid support, for example on the basis of corpusculaire matrix, Packed in a column and passing through the solution containing the protein on the column. Then associated protein elateroidea with the appropriate buffer. Typically, the protein is associated with the column under these physiological conditions, such as low ionic strength and a nearly neutral pH. The column is then washed to eleutherobia unbound impurities. Luterana associated protein is performed by changing the amount of the ionic strength or pH, for example, with 3M KSCN (group or gradient) or citrate buffer with low is ucirvine large number of megakaryocytes and platelets, which can be used, for example, to create cDNA libraries. Because platelets are directed to sites of damage, they can be regarded as the holders of wound healing and, under certain conditions, holders of pathogenesis. Therefore, the opportunity to teach molecular biology of platelets and megakaryocytes will give an understanding of how homeostasis, and clinical disorders of platelets. The proteins according to this invention provide improved means for producing libraries of megakaryocytes or platelets DNA.

Recombinant thrombopoietin when introduced into the animal or applied to the cultured cells of the spleen or bone marrow cause proliferation of megakaryocytes from precursor cells. Expansion (proliferation) of megakaryocytes and their precursors and maturation of megakaryocytes after the introduction of the TRO provides an opportunity to isolate the megakaryocytes with a high degree of purity and in sufficient quantity for isolation structures libraries mPHK and cDNA. Setting the dose of the solid and the mode of administration, sooner or fully Mature megakaryocytes and those that actively form platelets should selectively distributed from the primary spleen cells inimi, intermediate or late stages or megakaryopoiesis.

Applications of the resulting cDNA library a lot. Such libraries can, for example, be used for the identification and cloning of protein in a small amount, that has a certain value at different platelet dysfunction. The ease with which the megakaryocytes of the patient can be distributed, and its mRNA isolated for analysis, contributes significantly to the molecular descrii disease. Libraries are also sources cloning of new growth factors and other proteins with potential use in therapy. Useful proteins have been cloned platelets contain derived from platelet growth factor (Ross and other Cell 26:155 to 169, 1986); transforming growth factor (Miletich and other Blood 54:1015-1023, 1979); Roberts and Span, Growth Factors 8: 1-9, 199-3); growth factor endotheline cells derived from platelets (Miletich and other Blood 54:1015-1023, 1979) and PF-4 (Dua and others Mol.Cell.Biol. 7:898-904, 1987); Pont and other Blood 69:219-223, 1987). New growth factors can be identified by a detailed functional analysis of the library of expression of cDNA or gidratirovannym analysis at low strongevidence known sandopolis primers degeneration stored in regions of known growth factors. In addition, a systematic and complete DNA sequencing of the library creates a database megakaryocyte cDNA sequence. Such a database can be used in sequences in the application of various computer search algorithms.

The megakaryocytes, prepared as indicated above, can also be used to create a protein library. This protein library complementary to the cDNA library. Information about the amino acid sequence derived from a protein library, you can quickly carry out isolation of cDNA encoding the desired protein. Use of information about protein sequences when designing primers to isolate DNA, eliminate problems that arise when using the known methods of creating libraries, due to the relatively large number mPHK. The Association of libraries of protein and cDNA also simplifies labeled clone sequences that are of particular interest.

Protein library is created by selection of protein (whole proteins or their fragments) from megakaryocytes in accordance with known methods, and then in the separation of proteins using two-dimensional g separation using liquid chromatography high resolution (IHVR). The separated fragments are then investigated by the method of mass spectrometry. The obtained mass profile is studied on the basis of a database of protein sequence to a conclusion about the identity of the protein. Unidentified peptides can be sekvenirovan degradation of Edman.

Library cDNA and protein are valuable sources of new protein sequences of their coding. Platelets are important substances for healing wounds and under certain conditions pathogenesis. Have been identified and characterized many important trombotsitnoy proteins, including growth factors derived from platelets, transforming growth factor-growth factor endotheline cells derived from platelets, and trombotsitnoy factor 4. Identification and characterization of other trombotsitnoy proteins could be especially useful in explaining the processes underlying the healing of wounds and pathogenesis, and it is assumed that will give a lot of therapeutic substances and strategic methods.

As will be described in more detail below, genes of the human TPO localized on chromosome 3q26. This information, coupled with the gene sequence of human TPO (SEQ ID the x expression. Such violations include changes in sequences of promoters, leading to an increase or decrease in the level of expression, chromosomal translocations in the coding or non-coding regions, and the imposition of direct mutation and suppressor in the new regulatory sequences in the locus TRO. Diagnostic methods that may be used are well known in this field of science. For example, primers or hybridization probes of at least 5 nucleotides, and preferably from about 15-20 nucleotides in length can be constructed from the genomic sequence and used in the detection of chromosomal abnormalities or when measuring levels mPHK. In this field of science known many suitable methods for the detection and measurement, which include "southern" blotting, the chain reaction of polymerization (Mullis, U.S. Patent No. 4,683,202), chain reaction ligasale (Barany, PCR Methods and Application 1:5-16, Cold Spring Harbor Laboratory Press, 1991). For example, the DNA of the patient can be absorbed by one or more enzymes and converted to nitrocellulose for producing southern blot. Then this blot probing to detect major changes in the size of the fragment arising from mutations in the sequence recognition is s and abnormal sequences enable the creation of primers, which can be used to identify the abnormal gene (e.g., torn or translationmanager). The DNA of the patient amplificates chain reaction polymerization to detect characteristics of the amplified products of the normal gene or, in particular, genetic reorganization.

Further, the present invention is illustrated by the following non-limiting examples.

Example 1. Isolation of receptor DNA of the human MPL

Isoform of human MPL-P MPL-K receptor-encoding cDNA was isolated from human erythroid leukemia cells (Martin and Papadopoulo, Science 216:1233-1235, 1982) by the chain reaction reverse transcriptase polymerase using the primers of known sequence that encodes amino and carboxylate the end of receptors (Vigan and other OEWG.Natl.Acad.USA 89:5640-5644, 1992). Matrix cDNA of human erythroid leukemic cells was synthesized from poly-d(T)-selected poly(A)+RNA using primer ZC5499 (SEQ ID N0:3). Thirteen M1 poly(A)+RNA of human erythroid leukemic cells at a concentration of 1 mg/ml was mixed with 3 ml of 20 nmol/ml of the first receiver primer ZC5499 (SEQ ID NO:3). This mixture was heated at a temperature of 652) (5x SUPERSCRIPT TM buffer; GIBCO BRL, Gaithersbm'g, MD), 4 ml of 100 mm dithiothreitol and 3 ml of a solution of triphosphate of deoxynucleotide containing 10 ml of dATP, dGTP, dTTP and 5-d (Phatmacia LKB Biotechnology Inc., Piscataway, NJ). The reactive mixture was incubated at 45C for 4 minutes, then was added 10 ml of 200 U/ml reverse transcriptase RNase H- (reverse transcriptase SUPERSCRIPT TM; GTBCO BRL) in a mixture of RNA primer. This reaction was incubated at 45C for 1 hour and then at 50C for 15 minutes. To this reaction was added 60 ml of TE (10 mm Tris:Hcl, pH 80, 1 vV EDTA), followed by chromatography through a gel filtration column with 400 holes (chrOMA SPTN+ TE-400 tm); Clontech Laboratories Inc., Palo Alto, CA) to remove excess primer.

The first receiver of the DNA of human erythroid leukemic cells was used as the platelet by DNA amplification of human MPL receptor using primers corresponding to regilio, codereuse amino and carboxylate the end of the protein receptor (Wigan and others, ibid.). Each primer contains the restriction site of enzymatic hydrolysis, which contributes directed cloning of the amplified product of the th 10 ng trombotsitnoy cDNA, 50 Rola from each primer; 200 ml of each triphosphate of deoxynucleotide (Pharmacia LKB Biotechnology Inc.); 1 ml of 1 x PCR buffer (Promega Corp., Madison, WI) and 10 units of Taq polymerase (Roche Molecular Systems, Inc., Branchburg, NJ). Polymerase chain reaction was carried out for 35 cycles (1 minute at 95C, 1 minute at 60C and 2 min at 72With adding 1 extra second for each posleduyushchemu cycle), followed by incubation for 10 minutes at 72C.

cDNA-human MPL receptor were isolated by amplification of the polymerase chain reaction of cDNA of human erythroid leukemic cells by the way, identical to cDNA of the receptor MPL-P, described above, except that primer ZC5762 (SEQ ID NO:5) was replaced by ZC5742 (SEQ ID NO:6). PCR primer ZC5742-specific 3’ endings of the human cDNA MPL-K and is part of the restriction site Xho I to facilitate the cloning process.

The reaction products were extracted twice with phenol/chloroform (1:1), then once with chloroform and precipitated with ethanol. After absorbing the Eco RI and Xho I, these products were Fractionated on agarose gel with a low melting point 0.8% (PLA SEA is th matrix with agarti I was restored 1.9 thousand pairs of nucleotides (etc., ad) amplificatory product in accordance with the cDNA of human MPL-P receptor and 1.7, etc., N. in accordance with the cDNA of human MPL-K receptor (New England Biolabs, Inc., Beverly, MA), followed by ethanol precipitation. Acid cDNA were subcloned into the vector pBluescript @ SK+(Stratagene Cloning Systems, La Jolla, CA) for validation by sekvenirovaniya.

Example II. Isolation of cDNA malinovogo MPL-receptor

Mice C57BL/KsJ-db/db spleen were removed and immediately placed in liquid nitrogen. RNA was prepared from tissue of the spleen using isothiocyanate guanidine (Chirgwin etc. Biochemistry 18:52-94, 1979), followed by stage CsCl centrifugation. RNA poly+(A) spleen was isolated using oligo d(T) cellulose chromatography (Aviv and the lender, the OEWG. Natl.Acad.Aci.USA 69:1408-1412, 1972).

Seven and a half ml poly d(T) is selected from poly(A)+ RNA of mouse spleen at a concentration of 1.7 mg/ml were mixed with 3 ml of 20 nmol/ml of primer first barrel ZC66091 (SEQ ID NO:7) containing a restriction site Not I. This mixture was heated at 65C for 4 minutes and cooled on ice. Synthesis of cDNA the first barrel was initiated by the addition of 81 ml of 250 MM Tris-HCl, pH 8.3, 375 mM KCl, 15 mm MgCl2(5SUPERSCRIPT tm buffer; GIBCO RL), 4 ml of 100 mm lithotrite and 3 ml of a solution of triphosphate of deoxynucleotide containing 10 mm dATP, dGTP. dTTP and 5-methyl-dCTP (Pharmacia LCB Biotechnology Inc.) those were added 10 ml of 200 U/ml RNase H - reverse transcriptase (GIBCO BKL). The efficiency of synthesis of the first barrel was analyzed by conducting parallel reactions by adding 10 mCi32p-adCTP in the volume of reactive mixture, a multiple of 10 ml for labeling reactions for analysis. These reactions were incubated at 45C for 1 h, and then were incubated at 50C for 15 minutes. 32p-adCTP not included in the labeled reaction was removed by chromatography was carried out on a gel filtration column size 400 holes (chrOMA SPIN + TE-400 tm); Clontech Laboratories Inc.). Nucleotides that are not included in unlabeled reaction of the first barrel was removed by double precipitation of the cDNA in the presence of 8 mg of carrier glycogen, 2.5 ammonium acetate and 2.5 volume of ethanol. Unlabeled cDNA was translated in suspension in 50 ml of water for use for the synthesis of the second barrel. The length of the labeled cDNA of the first barrel was determined by agarose gel electrophoresis.

The synthesis of the second barrel was carried out on cDNA of the first barrel, provided that stimulated premirovanii on the first shaft of the second barrel will lead to the formation of hairpin loops) DNA. The reactive mixture was formed at room temperature and consisted of 50 ml of unlabeled cDNA of the first stem, 16.5 ml of water, 20 ml of buffer 5x polymerase I (100 mm is containing 10 mm triphosphate of deoxynucleotide, 3 ml of 5 mm NAD, 15 ml of 3U/ml E. coli DNA ligase (New England Biolabs Inc., Beverly, MA) and 5 ml 10/ml E. coli DNA polymerase I (Amersham Corp., Arlington Heights. IL). This reaction was incubated at room temperature for 5 minutes and then was added 1.5 ml 2 U/ml RNase H (GIBCO BRL). A parallel reaction in which the amount that is a multiple of 10 ml of synthesizing a mixture of the second barrel was marked by the addition of 10 MCI R-PAGE was used to control the efficiency of the synthesis of the second barrel. These reactions were incubated at 15C for 2 hours, and then were incubated for 14’5 minutes at room temperature. Not included in the labeled reaction 32p-adCTP was removed by chromatography through a gel filtration column size 400 holes (Clontech Laboratories, Inc.) before analysis by agarose gel electrophoresis. This unlabeled reaction was over two ekstragirovannymi with the extraction of phenol/chloroform and chloroform by ethanol precipitation in the presence of 2.5 M ammonium acetate.

Single DNA with hairpin structure was cut using nucleases bean Golden. The reactive mixture contained 100 ml of cDNA of the second barrel, 20 ml of 10buffer nuclease bean Golden (Stratagene Cloning Systems, La Jolla, CA), 16 ml itrace 10.5 U/ml) in buffer nuclease bean Golden. This reaction was incubated at 37 ° C for 15 minutes. The reaction was ended by adding 20 ml of 1 M Tris:Hcl, pH 8.0, followed by sequential extraction of phenol/chloroform and chloroform, as described above. After extraction the DNA was precipitated in ethanol and transferred into suspension in the water.

cDNA, newly translated into suspension, was blunted with all polymerase T4 DNA. cDNA, which was newly introduced in suspension in 190 ml of water, was mixed with 50 ml of buffer polymerase 5T4 DNA (250 mm Tris:Hcl, pH 8.0, mm KCl, 25 mM MgCl2), 3 ml of 0.1 M dithiothreitol in 3 ml of a solution containing 10 mm trifoliata of deoxynucleotide and 4 ml of 1U/ml polymerase T4 DNA (Boehringer Mannheim Corp.,, Indianapolos, IN). After incubation for 1 hour at 10The reaction was stopped by adding 10 ml of 0.5 M EDTA, followed by a series of extraction of phenol/chloroform and chloroform, as described above. DNA was chromatographically through Helou filtration column size 400 holes (Clontech Laboratories Inc., Palo Alto, CA) to remove small amounts of protein and removal of short cDNA, the length of which is less than -400 b(pair nucleotidase in suspension in 10 ml of water. As 32p-adCTP was used, the yield of cDNA was predicted order -2 mg on the initial value of 12.5 mg of platelets.

Eco RI adapters luigirules on the 5’ ends of the cDNA, giving the opportunity to spend cloning in lambda phage vector. The amount of cDNA that is a multiple of 10 ml (-2 mg) and 10 ml of 65 nmol/ml Eco RI adapter (Phannacia LKB Biotechnology Inc.) mixed with 2.5 ml of 10the ligase buffer (Promega Corp.), 1 ml of 10 mm ATP and 2 ml of 15 U/ml T4 DNA ligase (Promega Corp.). This reaction was incubated in the evening and overnight (about 18 hours) at a temperature gradient of from 0With up to 18C. Then the reaction was incubated in the evening and all night when 12C. Ended the reaction by adding 75 ml of water and 10 ml of 3 M Na acetate, followed by incubation at 65C for 30 minutes. After the incubation, the cDNA was extracted with phenol/chloroform and chloroform, as described above, and deposited in the presence of 2.5 M ammonium acetate and 1.2 volume of isopropanol. After centrifugation the precipitate cDNA were washed with 70% ethanol, dried in air and coming back into a balanced condition in 89 ml of water.

To facilitate directional cloning of cDNA in lambda phage vector, cDNA Ponza cDNA was inserted through the primer ZG6091 (SEQ ID NO:7). Absorption restricciones enzyme took place during the reaction, containing 89 ml cDNA, described above, 1 mm DTT (dithiothreitol) (10D buffer; Promega Corp., Madison, WI) and 1 µl of 12 U/μl NOt I (Promega Corp). Absorption was conducted with 37C for 1 hour. The reaction was ended by a series of ekstragirovanie phenol/chloroform and chloroform. cDNA was precipitated in ethanol, washed with 70% ethanol, dried in air and returned in suspension in 20 ml of 1x loaded gel buffer (10 mm Tris:Hcl, pH 8.0, 1 mm EDTA, 5% glycerol and 0.125% bromophenol blue).

Returned in suspension cDNA was heated at 65C for 5 minutes, cooled on ice and subjected to electrophoresis on 0.8% agarose gel with a low melting point (SEA PLAQUE GTG TM agarose, low melting point); FMC Corp.). Unused adapters and cDNA less than 1.6 Kb (thousand base pairs) in length was visiblility of the gel. The electrodes were changed signs, and cDNA was subjected to electrophoresis until then, until it begins to concentrate near the beginning of the path (stripes). The region of the gel containing concentrated cDNA was cut out and placed in the tube microcentrifuge, and determined the approximate amount Crespi heated to 65C for 15 minutes. After equilibration of the sample was added to the sample at 42With 10 ml of 1U/ml of agarose I (New England Biolabs, Inc.) and the mixture was incubated for 90 minutes for absorption of the agarose. After incubation, 40 ml of 3M Na acetate were added to the sample, and the mixture was incubated on ice for 15 minutes. This sample was centrifugals at 14,000g for 15 minutes at room temperature, which allowed to remove the unabsorbed agarose. cDNA found in the supernatant, was besieged in ethanol, washed in 70% ethanol, dried by air and returned in suspension at 37 ml of water for the reaction kinase to fosforistye legirovannykh Eco RT adapters.

In a solution of 37 ml of cDNA described above was added to 10 mlthe ligase buffer (Stratagene Cloning Systems) and the mixture was heated to 65C for 5 minutes. The mixture was cooled on ice and it was added 5 ml of 10 mm ATP and 3 ml of 10 U/ml of T4 kinase of polynucleotides (Stratagene Cloning Systems). The reaction was incubated at 37C for 45 minutes and ended up to 65C for 10 minutes, after which there was a series of extraction from Genoa, were washed with 70% ethanol, dried by air and coming back in suspension in 12.5 ml of water. The concentration of the phosphorylated cDNA estimated as approximately -40 mol/mm

The obtained cDNA was cloned into the lambda phage vector xll tm (Pharmacia LKB Biotechnology Inc.), prepared with Eco RI and Not I and the dephosphorylated. Ligation of cDNA to vector was carried out by reaction containing 2 ml of 20 f/ml of phage shoots.. tm ExCell, 4 ml water, 1 ml of 10the ligase buffer (Promega Corp.), 2 ml of 40 f/ml cDNA and 1 ml of 15 U/ml T4 DNA ligase (Promega Corp.). Ligation was performed at 4With in 48 hours. About 50% legirovannoi mixture was placed in the phage by sealing extract GIGAPACK II Gold (Stratagene Cloning Systems) in accordance with the directions of the vector. Resulting library contained over 1.5107independent recombinants with background levels besshabashnogo phage less than 1.5%.

The cDNA probe for human MPL-K receptor, marked R, was used to isolate cDNA of murine MPL receptor from a library of phage cDNA spleen. The cDNA library was planted on the SURE strain of E. coli cells. (Stratagene Cloning Systems) at a density of 40,000-50,000 PFU/Cup with a diameter of 150 mm Plague phage and the with the manufacturer's instructions. The treated filters were baked for 2 hours at 80With in a vacuum oven, followed by several washes at 70With wash buffer (0.25SSC, 0.25% SDS, 1 mm EDTA) and subjected to pre-hybridization overnight at 65In hybridizers solution (5SSC, 5 [denhardt's solution, 0.1% SDS, 1 mm EDTA and 100 mg/ml of warmed DNA denatured salmon sperm) in a hybridization oven (model HB-2; Techne Inc., Princeton, NJ). After prehybridization hybridizers solution is discarded and replaced with fresh hybridisation solution containing approximately 2106cpl/ml of human MPL-K DNA labeled R prepared with a set of tagging, commercially available (MEGAPRIME kit tm; Amersham Heights, ID). The probe was denaturirovannah at 98C for 5 minutes before it was added to hybridizers solution. Hybridization was performed at 65With during the night. Filters were washed at 55With wash buffer (0.25-SSC, 0.25SDS, 1 mM EDTA) and radioautography using intensifying screens techcast matrix weight of agar was restored in the regions, where was the bowl in accordance with the primary signals and usacialis in SM (0.1 M NaCl; 50 mM Tris:Hcl, pH 7.5, 0.02% gelatin) for eleutherobia phage to clean the virus plague. Was isolated seven purified from phage plaques that had the insert, hybridizers with probe human MPL-Receptor. Phagemid, which were in the phage tm ExCell, were restored by in vivo recombination system in accordance with the directions of the vector. The identity of the cDNA inserts was confirmed by sekvenirovanie DNA.

Isolated clones encode a protein, showing a greater degree sequenced identity of human MPL-P receptor and the recently announced murine MPL receptor (work Scody and others, EMBO J. 12:2645-2653, 1993). These clones fall into two classes that differ from the other three clones with deletions in the sequences encoding section 60 amino acid residues near the N-endings. cDNA encoding a protein without deletions, was named mouse cDNA Type I MPL receptor. cDNA for the Type II receptor had no sequence coding for residues of the receptor for Type I, 131 190 of SEQ ID NO:17. In addition, receptors Type I and Type II differed from the sequence of the murine MPL receptor (Skoda, etc is certain after amino acid residue 222, and replacement of the glycine residue in the serum in paragraph 241 (paragraphs correspond to the murine receptor Type I).

Acid cDNA of murine MPL receptor Type I and Type II were subcloned in the plasmid vector pHZ-1 for ekspressirovali in mammalian cells. Plasmids pHZ-1 is expressing vector that can be used to ekspressirovali protein in mammalian cells or systems IIT-broadcast frogs from mPHK, which was transcribed in vitro. Expressing unit pHZ-1 contains the murine promoter metallothionein-1, bacteriotherapy T7 promoter, flanked by banks of multiple clone containing a unique restricciones sites for insertion of coding sequences, terminator human growth hormone and terminator T7 bacteriophage. In addition, pHZ-1 contains the E. Coli replication to begin; gelbacterial beta-lactamase; expressing unit selective marker of the mammal containing the SV40 promoter, start, a gene of resistance to neomycin and the SV40 terminator of transcription. To facilitate directional cloning in pHZ-1 was used polymerase chain reaction with appropriate primers to create Eco RI site and Xho I site higher in comparison with the beginning of the launched erase in the mixture, containing 10 ml of 10x ULTMA tm buffer DNA polymerase (Roche Molecular Systems, Inc., Branchburg, NJ), 6 Ml of 25 mm MgCl2, 0.2 ml of a solution of triphosphate of deoxynucleotide containing 10 mm dATP, dGTP, dTTP and dCTP (Pharmacia LKB Biotechnology Inc.), 2.5 ml of 20 nmol/ml ZC6603 primer (SEQ ID NO:8), 2.5 ml of 20 nmol/ml ZG5762 primer (SEQ ID NO:5), 32.8 ml of water, 1 ml of log-phase bacterial culture, plasmids containing either murine MPL receptor Type I or Type II and 1 ml of 6 U/ml DNA polymerase (ULTMA tm polymerase; Roche Molecular Systems, Inc., Branchburg, NJ). In the reaction in accordance with the directions of the vector was used AmpliWax tm (Roche Molecular Systems, Inc.). Polymerase chain reaction was performed for 25 cycles (one minute at 95C, 1 minute at 55C and 3 minutes at 75C), followed by incubation for 10 minutes at 72C. Amplificatoare products were serially extracted with phenol/chloroform and chloroform, and then precipitated in ethanol in the presence of 6 mg of carrier glycogen and 2.5 M ammonium acetate. Precipitation was again transferred in suspension in 87 ml of water to which was added 10 ml of 10*H buffer (Boehringer Manneheim Corp.), 2 ml of 10 U/ml Xho I Eco RI (Boehringer Manneheim) and 1 ml of 40 U/ml Xho I (Boehringer Manneheim Corp.). c="https://img.russianpatents.com/chr/176.gif">C for 15 minutes and chromatographytandem through a gel filtration column size 400 holes (chrOMA SPIN + TE-400tm; Clontech Laboratories Inc.).

Insert the isolated receptor, described above, were doped in absorbed and dephosphorylating Eco RI and Xho I pHZ-1 vector. The alloying reaction contained 1 ml of 50 ug/ml pHZ-1 vector, 5 ml of 5 ng/ml insert cDNA, 2 ml of 10*buffer legasy (Promega Corp.), 11.75 ml of water and 0.25 ml of 4U/MA T4 DNA ligase (Stratagene Cloning Systems). Ligation was carried out at 10With during the night. Legirovannye acid DNA was transactionality in E. coli (MAX EFFICIENCY DH10B tm competent cells, GIBCO BRL). The validity of the inserts murine MPL receptor and human MPL-P receptor Type I and Type II was confirmed by DNA sequence. The resulting plasmid pSLmpl-8 and pSLmpl-9 were carriers of the murine MPL receptor Type I and Type II, respectively. Plasmids pSLmpl-44 was the carrier insert human MPL-P cDNA.

EXAMPLE III. The CREATION of CELL LINES F3 EXPRESSING MPL RECEPTORS

BaF3 - line pre-limpidly cells that are dependent on interleukin-3, taken from murine bone marrow (the work of Plastos and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevost and others, Mol.Cell.Biol.6:4133-4135, 1986), was maintained in complete medium (RPMI medium 1640 (JRH Bioscience Inc., Lenexa, KS), the stage is tigerbunny WEHI-3 cells (Becton Dikinson, Bedford, NA), 2 mM L-glutamine, 2-mercaptoethanol (final concentration 1:280,000), and PSN antibiotics (GIBCO BRL). Plasmids pSLmpl-8, pSLmpl-9 and pSLmpl-44, purified by cesium chloride, linearizability site Nde I to electroporation in BaF3 cells. For electroporation of cells F3 once were washed in RPMI medium 1640 and was again transferred in suspension in the medium BMPI 1640 at a density of cells 107cells/ml One ml of the newly translated into suspension cells F3 was mixed with 30 mg of DNA linearized plasmid and transferred into separate camera electroporation (GIBCO BRL). After 15 minutes incubation at room temperature, the cells were subjected to multiple shocks (800 mFAD/300 V; 1180 mFAD/300 V), coming from the apparatus electroporation (CELL-PORATOR tm; GIBCO BRL). After 5 minutes recovery time elektrooborudovanie cells were transferred to 10 ml of complete medium and were placed in the incubator for 15-24 hours (37C, 5% CO2). Then these cells were twisted and came back in suspension in 10 ml of complete medium containing 1600 mg/ml G418 and were inoculated with limited dilution in bowls for culturing tissue with 96 wells in order to isolate clones resistant to G-418. The expression of MPL receptor in cue transcript MPL receptor. It turned out that the cell line, designated BaF3/MPLR, I, expresses high levels of mRNA murine MPL receptor Type I and Type II, and was used in subsequent studies of MPL ligand in an air-conditioned environment transfection cells KSS 570. Line BaF3 cells expressing mPHK receptor Type II, was called BaF3/MPLR2.

EXAMPLE IV. The PRODUCTION of SOLUBLE MURINE MPL RECEPTOR

Expressing plasmids mammal that encodes a soluble murine MPL receptor Type I (pLEtapl-53), producyrovtsa by combining segments of DNA from pSLmpl-9 expressing plasmid mammal containing cDNA encoding murine MPL receptor full length described above, with a segment of DNA from pSLmpl-26 expressing the plasmids created for producing soluble murine MPL receptor Type I in bacteria. The cDNA segment encoding a soluble murine NPL receptor Type I was isolated by the PCR using primers ZC6704 (SEQ ID NO:10) and ZC6703 (SEQ ID NO:11), where the matrix was used plasmids pSLmpl-9 receptor full length. To facilitate directional cloning primers ZC6704 and Z6703 included Eco RI and Xho I restricciones sites accordingly at their ends 5’. Also primer ZC6703 encodes the consensus sequence R. Proc.Natl.Acad. Sci. USA. 86:558-562, 1989). PCR was performed in a mixture containing 10 ml of 10buffer ULTMA DNA tm (Roche Molecular Systems, Inc.), 6 ml of 25 mm MgCl2, 0.2 ml of a solution of triphosphate of deoxynucleotide containing 10 mm dATP, dGTP, dTTP and dCTP (Pharmacia LB Biotechnology Inc.), 11 ml of 4.55 nmol/ml primer ZC6704 (SEQ ID NO:10), 21 ml of 2.43 nmol/ml primer ZC6703 (SEQ ID NO:11), 50.3 ml water, 1 ml 50 ng/ml pSLmpl-9 absorbed Hind III and Xba and 1 ml of 6U/ml polymerase HKULTNA tm (Roche Molecular Systems, Inc.). In the reaction in accordance with the instructions of the seller used AmpliWaz tm (Roche Molecular Systems Inc.). Polymerase chain reaction was carried out in 3 cycles (1 minute at 95C, 1 minute at 50C and 2 min at 72C), after which was 11 cycles at elevated requirements hybridization (1 minute at 95C, 30 seconds at 55C and 2 min at 72C), followed by incubation for 10 minutes at 72C. Amplificatory product then was extracted with phenol/chloroform and chloroform, followed by chromatography through a gel filtration column size 400 holes (Clontech Laboratories, Inc.). PRC product was precipitated in ethanol in presence state is s. To 16 ml returned in suspension of PCR product was added 2 ml of 10buffer P (Boehringer Mannheim Corp.), 1 ml of 10U/ml Eco RI (Boehringer Mannheim Corp.) and 1 ml of 40 U/ml Xho I (Boehring Mannheim Corp.). Absorption occurred at 37C for 1 hour. This absorption was finished by heating to 65C for 15 minutes, then was cleaned by 0.7% gel agarase with a low melting point. The recovery of fragments from agarose with a low melting point was carried out by absorption of the gel matrix with beta-agarose I (New England Biolabs).

Thus obtained PCR product encodes the extracellular region of the N-terminal murine MPL receptor Type I (residues 27-480 of SEQ ID NO:17). In the absence of TRANS-membrane region of the proposed receptor (residues 483-504 of SEQ ID NO:17), it is assumed that the downregulation of protein were separated in the presence of a signal peptide. Murine MPL receptor Type II encoding cDNA, was obtained using the PCR conditions described above, except that, as the matrix was used pSLmpl-8. The validity of both fragments of the receptor is confirmed by sekvenirovanie DNA.

Soluble murine MPL receptor Type I and Type II were cloned in the battle modification Rome (work and other Graab EMBO J. 3:2437-2442, 1984), bacterial expressing vector absorbed to accommodate recombinant protein in perepleteny period. pOmpA2-5 was constructed by replacing the sequence of the 13 base pairs between sites Eco RI and You N Rome synthetic sequence from 42 pairs of nucleotides. The sequence was created by annealing two complementary oligonucleotides of 42 nucleotides (ZC6707, SEQ ID NO:12; ZC 6706, SEQ ID NO:13), which formed the sticky ends of the DNA, making it easier for directional cloning in Roma, absorbed Eco RI and You HI. Within the inserted sequence is the Xho site I built with regard to bacterial leader sequence and the mouse soluble MPL receptor encoding cDNA described above, as well as a built-in path (section) of the 6 histidine codons located in the 3’ site Xho I, which allows to purify the recombinant protein by affinity chromatography chelate metals (work Hochuli and other Bio/Technol.6:1321-1325 1988). For a sequence that encodes a histidine tract, followed built target codon. The validity Rome-5, pSLmpl-26 and pSLmpl-27 was confirmed by sekvenirovanie DNA.

pLDmpl-53, expressing plasmids mammal, producing soluble is the vector of pHZ-200 (pHZ-1, in which the sequence reductase dihydrofolate was replaced with a gene resistant to neomycin). The cDNA fragment Eco RI/Bam HI from 1164 pairs of SIml-9 was replaced by the signal sequence of a mammal, deliciouso during construction expressing bacterial plasmid pSLmpl-26. Fragment Bam HI from 416 base pairs of pSLmpl-26 provided the coding sequence of the end carboxide part soluble MPL receptor, region labeling kinase, a tract of polyhistidine and terminator broadcasting. Two fragments were cleaning gel and were cloned in sites Eco RI/Bam HI from pBluescrilt R KS+ (Stratagene Cloning System) to obtain plasmid pBS8.76LD-5. The correct orientation of the fragment Bam HI abstracted from pSLmpl-26 of 416 base pairs relative to the slice Eco RI/Bam HI abstracted from pSLmpl-9 from 1164 nucleotides in pBS8.76LD-5, was determined by PCR using primer ZC6603 (SEQ ID NO:8) and ZC 6703 (SEQ ID NO:11). The website Xba I inside the poly-linker sequence pBS8.76LD-5 gave the ability to cut recreated cDNA of the receptor, a fragment of the Eco RI/Xba 1.5 thousand pairs of nucleotides for cloning in pHZ-200, after absorbing the vector Eco RI and Xba I. the resulting expressing plasmids mammal, pLDmpl-53, prepared Pirovano in 570 cells KSS using the method of deposition of sodium phosphate. After 5 hours the cells were operated 15% glycerol for 3 minutes to facilitate the uptake of DNA. During the night added fresh growth medium. The next day, cells were split at various concentrations of the solution and was added to the electoral medium containing 3 mm methotrexate. After about 2 weeks became visible discrete resistant methotrexate colony. These colonies were either grouped or remained in certain clones.

Spent medium was immediately tested for the presence of soluble protein MPL receptor. Protein soluble MPL receptor were isolated through interaction tract poly-histidine present at carboxide the end of the protein from the resin of a metal chelate containing devoid of movement Ni2+(HISBIND tm; Novagen, Madison WI).The environment, culture, free from serum from a group pLDmpl-53 passed over the resin and boundary protein was sotirova 1M imidazole. Analysis of SDS-PAGE revealed a single band at -67 kilodalton (kDa). This protein was subjected to analysis using amino acids from the N-end and it was confirmed that this mouse MPL receptor.

Soluble murine MPL receptor was purified from transfectants Vnesenii soluble receptor has been denied the motion on CNBr-activated SEPHAROSE matrix tm 4B (Pharmacia LKB Biotechnology, Inc.), especially if it has the manufacturer's instructions, and used for affinity purification of aktivnosti MPL in the conditioned media of cells 24-11-5. Affine matrix was Packed in a column HK (Pharmacia LKB Biotechnology Inc.). The air-conditioned environment of 24-11-5 cells is concentrated on the cut of the hollow fiber membrane of 10 Kd (A/G Technology Corp., Needham, NA) and sank to the bottom of the affinity column MPL receptor at a flow rate of 1 ml/minute. This column were washed in phosphate saline solution containing 0.5 M NaCl and 0.01% sodium azide. The activities of the MPL was eleutheropolis from the column with 3M potassium thiocyanate (Sigma Chemical Company, St.Louis, MO) at a flow rate of 0.5 ml/min. The potassium thiocyanate was removed by dialysis against phosphate saline solution. Active fractions were detected using MTT proliferation analysis (discussed in Example VII).

EXAMPLE V. ISOLATION AND characterization of a CELL LINE EXPRESSING the LIGAND MPL RECEPTOR

Cells BaF3/MPLR1.1 are IL-3 dependent cells expressing stably transfectional murine MPL receptor Type I. Were invented mutagenesis and scheme selection to isolate cell lines expressing the ligand MPL receptor with cells mutagenicity is sup>6cells BaF3/MPLR1.1 was precipitated and washed in GM (medium RPMI 1640, supplemented with 2-mercaptoethanol (1:240,000 - final concentration), 2 mm L-glutamine, 110 mg/ml sodium pyruvate, 50 mg/ml G418 and 10% nonactivated fetal bovine serum). These cells were again transferred into suspension in 2 ml of GM containing 0-15% (v/v) mutagenic 2-ethylmethanesulfonate (EMS), and were incubated for 2 hours at 37C. After incubation the cells were washed once in PBS and once in GM and was sown on the bowl 10 cm with a density of about 40,000 cells/ml in GM supplemented with 5% WEHI-3 conditioned medium (Becton Dickinson Labware, MA) as a source of IL-3. These cells were given a recovery period of seven days and isoberbinia was held at 37With 5% CO2before selection for independent growth of IL-3. After a period of recovery culture compacted viable cells. Cells were washed with GM and was cultivated in GM in the absence of air-conditioned environments WEHI-3. After 11 days of selection were observed in a small number of viable cells. The density of viable cells in culture, independent of IL-3 were predicted to equal to 250 cells/ml One ml independent of>additionalone environment of the above-mentioned independent of IL-3 cells F3/MPLR1.1 were tested for proliferative activity relative to cells BaF3/MPLR. It turned out that air-conditioned environment of the 19 independent of IL-3 groups have growth activity in the sample to be tested (described in Example VII). A positive environment were analysed for proliferative activity in the presence of 2 mg/ml rat antimisting IL-3, antimisting IL-4 or in the presence of both neutralizing tel (Pharmingen, San Diego, CA) to identify mutants, independent of the growth of IL-3 expressing these cytokines. (In the previous experiments, it was found that the BaF3 cells also responded to IL-4). Only in the conditioned medium of cells from the bowl No. 11 (the cell with the designation "24-11) was detected activity, which was kind of balanced out by antibodies to IL-3 and IL-4.

Scheme of mutagenesis and selection, described above, was applied to five other BaF3/MPLR1 clones (BaF3/MPLR1 clones No. 4696126 15 and 18, designated as BaF3/MPLR1-4,-9,-12,-15 and-18 respectively). It turned out that the seventeen isolates (strains) were air-conditioned environment that stimulated the proliferation of BaF3 cells/MPLR1. The activity of all these environments were neutral is ü.

Proliferative activity of conditioned media from the group 24-11 was characterized in detail. Group 24-11 was divided into 19 subgroups, and air-conditioned environment, were re-tested for activity. All 19 sub-groups (i.e. 24-11-1 on 24-11-19 inclusive) stimulated the proliferation of BaF3 cells/MPLR1-independent growth of IL-3 in the absence of exogenous IL-3. This activity was not suppressed by neutralizing antibodies to IL-3 or IL-4, or a combination of these antibodies.

Conducted two experiments to determine the features of the activity 24-11. Conditioned medium was analyzed for proliferative activity in the control BaF3 cells that do not Express the receptor MPL. In the absence of exogenous IL-3 proliferation control BaF3 cells was not observed in air-conditioned environments, none of the nineteen 24-11 subgroups. In the second experiment, the proliferative activity was investigated on the suppression of purified soluble MPL receptor. Cells BaF3/MPLR1 was cultivated in medium GM, to which was added a 5% air-conditioned environments 24-11. To each sample was added to the soluble murine MPL receptor Type I to a final concentration of 0.0, 0.625, 1.25, 2.5 or 5.0 mg/ml. the Results were affected by four days when anal the van at a concentration of 0.625-1.25 mg/ml soluble MPL receptor. The concentration of soluble receptor, which completely stop the activity, not acted to stimulate cells BaF3/MPLR1 with IL-3 or IL-4. The results showed that soluble MPL receptor can compete with the hypothesis that 24-11 cells expressed the MPL ligand of the receptor.

Clones taken away from the cells 24-11, isolated seeding at limiting concentrations of solutions. One clone, designated 24-11-5 No. 3, showed a high level of proliferative activity in air-conditioned environments in relation to the group 24-11. Proliferative activity was equal to 1:2000 dilution of conditioned media from cells WEHI-3 (Becton Dickinson Labware).

EXAMPLE VI. CONSTRUCTION of cDNA LIBRARY 24-11-5 №3

Total RNA was prepared from -2.7108cells 24-11-5 No. 3 with isothiocyanate of guanidine, followed by centrifugation in CsCl (work Chirgwin and others, ibid.). Poly(A)+ was isolated using the kit to isolate OLIGOTEX-dT-mPHK (Qiagen Inc., Chatsworth, CA) according to the manufacturer's instructions.

Acid cDNA first stem cell 24-11-5 No. 3 was synthesized in 4 parallel reactions. Each Roccia contained 7 ml d(T)-selected poly(A)+24-11-5 No. 3 RNA at the end of the This mixture was heated at 65C for 4 minutes and cooled on ice. Synthesis of cDNA the first barrel was initiated by adding 8 ml of a buffer of the first barrel (5x buffer SUPERSCRIPT tm; GIBCO BRL), 4 ml of 100 mm dithiothreitol and 2 ml deoxynucleotide triphosphate containing 10 mm dATP, dGTP, dTTP and 5-methyl-d (Pharmacia LKB Biotechnology Inc.) in a mixture of RNA primer. The reactive mixture was incubated at 45C for 4 minutes, followed by adding 10 ml of 200 U/ml reverse transcriptase RNase H (GIBCO BRL). The efficiency of synthesis of the first barrel was analyzed in a parallel reaction by adding 10 MCI of 32p-adCTP in the amount divisible by 10 ml of one of the reactive mixtures for the labeling reaction for analysis. These reactions were incubated at 45C for 1 hour, followed by incubation at 50C for 15 minutes. Unused 32p-adCTP in the labeled reaction was removed by chromatography on a gel filtration column size 400 holes (Clontech Laboratories). Unlabeled reaction of the first barrel were grouped and unused nucleotides were removed by double precipitation of the cDNA in the presence of 32 mg of carrier glycogen, 2.5 M ammonium acetate and 2.5 volume of ethanol. Unlabeled cDNA newly pens the first barrel was determined by gel-electrophoresis agarose.

The synthesis of the second barrel was carried out on cDNA of the first shaft under the condition that the synthesis of the second shaft driven premirovanii the first barrel, will lead to the formation of loops of DNA. There were three independent parallel reactions of the second barrel. Each reaction of the second barrel contained 48 ml unlabeled cDNA of the first stem, 16.5 ml of water, 20 ml of buffer polymerase I (100 ml Tris:Hcl, pH 7.4, 500 mm KCl, 25 mm MgCl2, 50 mM (NH4)2SO4), 1 ml of 100 mm dithiothreitol, 1 ml of a solution containing 10 mm deoxynucleotide triphosphate, 3 ml of 5 mm beta-NAD, 1 ml of 3U/ml E. coli DNA ligase (New England Biolabs Inc.) and 5 ml of 10 U/ml of E. coli DNA polymerase I (Amersham Corp.). The reaction was conducted at room temperature and were incubated at room temperature for 5 minutes, after which was added 1.5 ml of 2 U/ml RNase H (GIBCO BRL). 10 ml of one of the reactions of synthesis of the second barrel was marked by the addition of 1 MCI of 32-p-adCTP to regulate the efficiency of synthesis of the second barrel. These reactions were incubated at 15°C for 2 hours, followed by incubation for 15 minutes at room temperature. Unused labeled reaction 32p-adCTP was removed by chromatography through a gel-filtration column with a size of 400 holes (Clontech Laboratories) p is with phenol/chloroform and chloroform, followed by precipitation in ethanol in the presence of 2.5 M ammonium acetate.

Single DNA loop structure was cut using nucleases bean Golden. The reactive mixture contained 100 ml of cDNA of the second barrel 20 ml 10x buffer nuclease bean Golden (Stratagene Cloning Systems), 16 ml of 100 mm dithiothreitol, 48 ml of water, 10 ml diluting buffer nuclease bean Golden (Stratagene Cloning Systems) and 6 ml of 50 U/ml nuclease bean Golden (Promega Corp.). The reaction was incubated at 37C for 30 minutes. The reaction was ended by adding 20 ml of 1 M Tris:Hcl, pH 8.0, then walked sequential extraction of phenol/chloroform and chloroform, as described above. After extraction the DNA was precipitated in ethanol and re-translated in suspension.

Newly translated into suspension cDNA was blunted by T4 DNA polymerase ends. cDNA, which was translated into suspension in 188 ml of water, was mixed with 50 ml of 5x buffer, polymerase, T4 DNA (250 mm Tris:model HC1, pH 8.0, 250 mm RCl, 25 mM MgCl2), 3 mm deoxynucleotide triphosphate and 5 ml of 1U/ml polimery T4 DNA (Boehringer Mannheim Corp.). After incubation for 30 minutes at 15The reaction was over the house, as was described above. DNA was chromatographically through the gel filtration column with a size of 400 holes (Clontech Laboratories Inc.) removal of a small amount of protein and removal of short DNA whose length is less than -400 pairs of nucleotides. DNA was precipitated in ethanol in the presence of 10 mg of carrier glycogen and 2.5 M ammonium acetate and was again transferred in suspension in 15 ml of water. On the basis of using 32p-adCTP the size of the resulting DNA is projected to equal -8 mg of the initial amount of mRNA matrices equal to 40 mg.

Adapters Eco RI were legirovanyh on the 5’ ends of the cDNA described above, in order to give the opportunity to spend cloning in expressing vector. 10 ml of cDNA (-5 mg) and 21 ml mol/ml adapter Eco RI (Pharmacia LKB Biotechnology Inc.) mixed with 4 ml of 10the ligase buffer (Proinega Corp.), 3 ml of 10 mm ATP and 3 ml of 15 U/ml T4 DNA ligase (Promega Corp.). The reaction was incubated overnight (-48 hours) when 9C. the Reaction was ended by adding 140 ml of water, 20 ml of 10H buffer (Boehringer Mannheim Corp.) and were incubated at 65C for 40 minutes. After incubation cDNA was extracted with phenol/chloroform and chloroform, as described above, and deposited at p is anolon, dried air and returned in vzveshennoe state in 89 mg of water.

To facilitate directional cloning of cDNA in expressing vector cDNA was absorbed Xho I, which led to the fact that the cDNA formed a 5’ Eco RI adhesive end and a 3’ Xho I sticky end. Pre was introduced restrictions website Xho I (3 konza cDNA using primer ZC6172 (SEQ ID NO:14). Absorption restricciones enzyme was carried out in a reaction mixture containing 89 ml cDNA described above, 10 ml of 10N buffer (Promega Corp.) and 1.5 ml of 40U/ml HSA I (Boehringer Mannheim Corp.). The absorption was carried out at 37C for 1 hour. The reaction was ended serial ekstragirovannymi phenol/chloroform and chloroform, and chromatography through a gel-filtration column with a size of 400 holes (Clontech Laboratories Inc.).

cDNA was precipitated in ethanol, washed in 70% ethanol, dried in air and coming back in suspension in 20 ml 1loaded with gel buffer (10 mm Tris:Hcl, pH 8.0, 1 mm EDTA, 5% glycerol and 0.125% Bromphenol blue. Returned in suspension cDNA was heated to 65C for 5 minutes, cooled on ice and subjected to electrophoresis on mesnie adapters and cDNA length of less than 0.5 thousand nucleotides were cut from the gel. The electrodes were changed signs and cDNA was subjected to electrophoresis as long as she does not concentrate near the beginning of the period. The region of the gel containing concentrated cDNA, were cut and placed in the tube microcentrifuge, and determined the approximate volume of the gel slice. The amount of water, about three times the volume of the gel slice (300 ml), was added to the tube, and the agarose was melted by heating to 65C for 15 minutes. After reaching the equilibrium state of the sample at 45With added 5 ml of 1U/ml beta-agarose I (New England Biolabs, Inc.) and the mixture was incubated for 90 minutes at 45For acquisitions agarose. After incubation for 40 ml of 3 M Na acetate was added to the sample and the mixture was incubated on ice for 15 minutes. This sample was centrifugals at 14,000g for 15 minutes at room temperature to remove unabsorbed agarose, followed by chromatography through a gel-filtration column with a size of 400 holes (Clontech Laboratories). cDNA was precipitated in ethanol, washed in 70% ethanol, dried in air and returned in suspension in 70 ml of water for PRS 10 ml of 10buffer ligase (Stratagene Cloning System), and the mixture was heated to 65C for 5 minutes. The mixture was cooled on ice, and 16 ml of 10 mm ATP and 4 ml of 10 U/ml T4 polynucleotide kinase (Stratagene Cloning Systems) were added. The reactive mixture was incubated at 37C for 1 hour and the reaction was ended by heating 65C for 10 minutes, followed by sequential extraction with phenol/chloroform and chloroform. Phosphorylated cDNA was precipitated in ethanol in the presence of 2.5 M ammonium acetate, was washed with 70% ethanol, dried in air and returned in suspension in 10 ml of water. The concentration of the phosphorylated cDNA was estimated as -40 f/ml.

Expressing pDX vector mammal (discussed in U.S. Patent No. 4,959,318, Figure) was modificirovana for perception 24-11-5 No. 3 cDNA that was synthesized from the ends of the Eco RI-Xho I. Endogenous Sal website at pDX was eliminated by absorption of plasmid with Sal I and recircularisation plasmid, which followed after the break sticky ends Sal I polymerase T4 DNA. Recycled plasmids were absorbed Eco RI and it was luigirules short polylinker sequence of two complementarities at pDX.ES consisted of sites Eco RI and Sal I, that was simplified directional cloning of cDNA 24-11-5 synthesized with Eco RI-Xho I.

The cDNA library plasmid was created by legirovaniem cDNA Eco RI-Xho I 24-11-5 in pDX.ES, absorbed Eco RI/Sal I. the ligation Mixture was elektroporcelana in E. coli (competent cells ELECTROMAX DH10B tm; GIBCO BRL, Gaithersburg, MD) using a pulse controller and 0.2 cm cuvette (Bio-Rad Laboratories, Hercules, CA), using 0.2 KB, 400 Ohms and 25 mFAD. These cells were diluted in broth, Luria and were incubated at 37C for 45 minutes, after which was added 0.75 ml of 50% glycerol. Transfection cells were liquation and stored at -70With to use. 80 f cDNA generated more than 700,000 independent recombinant plasmids.

EXAMPLE VII. EXPRESSIVE RESEARCH LIBRARY cDNA 24-11-5 ON MPL ACTIVITY

The cDNA library 24-11-5 No. 3 was sown on about 2 thousand agar cups with a diameter of 10 cm with the broth Luria, to which was added 100 mg/ml ampicilin. The sowing density was equal to 200 to 250 bacterial colonies on the Cup. DNA plasmid for transfection in KSS 570 cells were prepared from each bacterial bowl with cleansing DNA resins MAGIC MINIPREPS tm (Promega Corp.), in accordance with the manufacturer's instructions. what the Idov cDNA 24-11-5 No. 3, each of which contained approximately 200-250 cDNA clones were transactionality in KSS 570 cells using recipe 3:1 liposome 2,3-dialerace-N-{2(permineralized)ethyl}-N,N-dimethyl-1-propanenitrile and valeriefitzgerald in water (LIPOFECTAMINE tm; GIBCO BRL). 20 ml of 30 ng/ml of DNA was added to 20 ml of the solution of LIPOFECTAMINE tm, diluted in a ratio of 1:10, and incubation was performed at room temperature for 30 minutes. After incubation 160 ml of media free of serum (Hams F12: Dulbeccos MEM (1:1) supplemented with 2 mm L-glutamine, 0.11 mg/ml sodium pyruvate, 5 mg/ml insulin, 5 mg/ml of fetuin, 10 mg/ml transferrin, 2 ng/ml monoxide IV selenium and 25 mm HEPES buffer) in a mixture of DNA/LIPOFECTAMINE tm. Then it was transferred to a 42-locovei tetrazinni the microplate containing -100,000 KSS 570 cells. These cells were incubated at 37With 5% CO2within 4 hours, after which was added 200 ml KSS Media Growth (Environment Inglés, modified Dulbecco to which you have added 2 mm L-glutamine, 0.11 mg/ml sodium pyruvate, 5% fetal calf inactivated serum and 100x PSN antibiotics (GIBCO BRL). These cells were incubated for 16 hours. The medium was removed and replaced by 0.5 ml of fresh Medium Growth KSS, which condizionale MPL in air-conditioned environment of the library, transactionally KSS 570 cells, used the analysis of cell proliferation. 100 ml of conditioned media was added to 100 ml of 10 6/ml washed cells BaF3/MPLR1.1 in environments RMPI 1640 (JRH ioscience Inc., Lenexa, KS) supplemented with 2 mm L-glutamine, and PSN antibiotics (GIBCO BRL), 0.00036% 2-mercaptoethanol and 10% heated fetal inactivated calf serum. Subjects the cells were incubated for 3 days at 37With 5% CO2prior research on the proliferation.

Cell proliferation in the presence of the MPL was determined quantitatively using calorimetric analysis based on the metabolic decomposition of 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetryzoline bromide (MTT) (Mosman, J. Immunol.Meth.65:55 to 63, 1983). 20 ml of 10 mg/ml solution of MTT (Polyscience, Inc., Warrington, PA) was added to 100 ml of the tested cells BaF3/MPLR1.1, and these cells were incubated at 37C. After 4 hours was added 200 ml of 0.04 N Hcl in isopropanol, the solution was mixed, and the adsorbent sample was considered at 570 nm on a plate reader tablet model EL320 ELISA (Bio-Tek Instruments Inc., Highland Park, VT).

One of the plasmid bands were positive, denoted T, transactionality cells KSS 570. The supernatant from the transfectants gave a positive signal PR is not due to IL-3 and IL-4.

Plasmids from positive groups was used to transform E. coli DH10B, and the cells were inoculated (42 bowl with about 15-20 colonies on the bowl 10 cups with about 90 colonies on the bowl and 8 cups with about 250 colonies on the bowl). Each bowl was made print (replica) and remained atC. Colonies were schedules with the original bowls and freely prerastali in liquid culture for another few hours, then got ready DNA.

Plasmid DNA from subgroups were transactionality in KSS 570 cells and cell supernatant was collected and analysed as described above. About two hours later one subgroup (No. 22) was positive in the microscopic studies (the cell is elongated). A few hours later two more subgroups (No. 19 and No. 28) was also positive. The remaining supernatant from each positive group were analyzed and compared with the control BaF3 cells and found that it had no activity. In addition, it was found that the activity of these three positive subgroup was suppressed soluble MPL receptor Type I.

Prints bowls of the three positive subgroups grew back in a few hours. Then individual (separate) colonies of sousou at 37Since, then, were prepared DNA by the method described above. Plasmid DNA was transactionality in KSS 570 cells and the supernatant was grown about 10 hours later and analyzed for the activity. After 1 hour one clone (designated T-19-215 corresponding to the subgroup 19) were positive. This clone were inoculated strip again for single colonies. DNA was prepared from 12 colonies and transactionality in KSS 570 cells. All 12 transfectants later turned out to be positive in the study. DNA from one of the 12 positive colonies were transformed into E-Li DH5a. The plasmid was designated pZGmp1-1081. This transformant was depositional February 14, 1994 at the American Meeting of the types of Crops, 12301 Parklawn Drive, Rockville, MD, under catalogue number 69566.

The nucleotide sequence of cDNA encoding a hematopoietic protein (thrombopoietin), was determined (SEQ ID NO:1). Analysis of the encoded amino acid sequence (SQ ID NO:2) showed that the amino end of the Mature protein is an amino acid residue 45. Two methioninol codon in paragraphs 105 and 174 of SEQ ID NO:1 was the initiation codons, and the primary site of initiation is assumed in paragraph 174.

EXAMPLE VIII. HEMATOPOIETIC ACTIVITY Rekordowa period were cultured in 25 ml buffer CATCH (99 mg teofilina, 0.75 g of sodium citrate, 75 mg of adenosine, 20 ml of 10balanced saline solution, free of Ca++Mg++, 200 ml dH2O; pH 7.4). Cells were suspended in a single cell suspension by unearth from 25-ml pipette. This amount had grown to 50 ml with buffer a CATCH, and the cells were precipitated by centrifugation at a speed of 1000 rpm for 7 minutes. The residue was again transferred in suspension in 20 ml of buffer a CATCH and were incubated in a flask for culturing tissue Kzt75 during the first cycle of plastic adhesion at 37C for 2 hours. Neadgezivnye cells were collected by centrifugation at a rotation speed of 1000 rpm for 7 minutes, allowing to precipitate the cells. The residue was again transferred in suspension in 15 ml of alpha-MEM+ 10% FBS (+L-glutamine the N and PSN antibiotics) and incubated in a flask Kzt75 for the second cycle of plastic adhesion, as described above for the first cycle. After the final centrifugation and placed in suspension cells were counted. Half ml of all cells in 576,000 cells/ml were seeded in 24-Lunnoye bowl for culturing tissue together with trial by media control is berbania at 37Since the cells were collected and stained as described above.

150 ml of cells were collected from the control wells in the processing in standard air-conditioned environment. 50 ml of cells were collected from wells treated with conditioned medium from KSS cells, transfection pZGmpl-1081. These samples were mixed and made standard mikroskopicheskie slides.

These slides were preserved in 100% methanol, then was filled with a solution of Wright in 1:1 ratio (0.5 g dye Wright in 300 ml of methanol)/N2O for 6 minutes, washed with water and dried. Then the slides were flushed of Giemsa (Sigma Chemical Corp.) in the buffer Sorensen (2.28 g KN2RHO4/2&38 g Na4in 250 ml of N2O), washed in water and dried.

After uregulirovania used volumes of sample environment BHK/pZGmpl-1081 consisted of 120 megakaryocytes 150 ml compared with 9 megakaryocytes in 150 ml of test medium. In addition, the megakaryocytes in the treated experimental sample observed in the microscope, were significantly larger than control cells, and had a much more colored polynucleotide.

The air-conditioned environment mutant BaF3/MPLR1.1 line 24-11-5 No. 3 was collected in the absence of serum and concentrated 20-SK the brain and weighed in Environments Dulbecco, modified Claims (GIBCO BRL)+ 15% fetal calf serum (FCS). After translation in suspension were counted nucleotide cells and were inoculated at 75,000 cells/ml and 0.9 ml/Cup in a medium containing 50% methylcellulose, 15% FSC and 10% BSA and 0.6%PSN (semi-solid medium) in bowls for culturing the tissue in a volume of 1 ml was Added a variety of air-conditioned environment and control samples to a final volume of 1 ml Cups were incubated at 37C/5% CO2within 6 days, and then examined under a microscope for counting colonies granulocytic/ mikrofalowe colonies. It turned out that bowl, incubated in the presence of air-conditioned environment 24-11-5 No. 3, had a weak GMCSF-like activity, expressed in quantity of 25, compared to zero in the case of a negative control sample and 130 for bowls, stimulated positive control (medium prepared by treatment of the spleen Phytolacca American (PWMSCM) method incubation crushed mouse spleen during 1 week in the presence of mitogen, Phytolacca American (obtained from Boehringer Mannheim, Indianapolis, IN) + 2 units/ml erythropoietin).

From mouse femur were taken substance of bone Ki were calculated and were cultivated in semi-solid medium, as was described above. These cells were used to test the colony of megakaryocytes, creating activity of the protein encoded by the insert pZGmpl-1081.

Group KSS 570 cells stably transfecting with pZGmpl-1081, was cultivated in the absence of serum, and was going to air-conditioned environment. This air-conditioned environment was tested individually and in combination with the environment spleen treated Phytolacca American, with recombinant murine IL-3, IL-4 (Genzyme Corp., Cambridge, MA), IL-11 (Genzyme Corp.) different combinations of these factors. PWMSCM was used as a positive control. As a negative control were used reconditionnement environment cultivation.

Subjects or control samples were added to the cultures of the substance of the bone marrow to add up to a volume of 1 ml Bowls were incubated for 6 days at 37With 5% CO2, then examined under a microscope to count megakaryocytic colonies. The results are shown in Table 4. Summarizing, we can say that air-conditioned environment BHK/pZGmpl-1081 showed the presence of megakaryocyte colony forming activity, which is enhanced in the presence of early acting factors to levels mean the awn air-conditioned environment BHK/pZGmpl-1081 was investigated in mice. Was collected serum-free medium and concentrated in 5 folds using 10 Kd with the help of the device to filter cutoff (Amicon Inc., Beverly, NA). Control (non-condensing) environment focused in the same way. 6 BALB/c mice (Simonsen Laboratories Inc., Gilroy, CA) were processed seven daily abdominal injections of 0.5 ml of either control or air-conditioned environment. A blood sample was taken on the following days: 0. 3 and 7, and it has been estimated platelets. The results are shown in Table 5, showed that conditioned medium from BHK cells/pZGmpl-1081 had thrombopoetin activity.

EXAMPLE IX. ISOLATION of HUMAN TROMBOTICHESKOE GENE

Amplificatory of Enoteca human LAMBDA light FIX R (Stratagene Cloning Systems) has been studied extensively on the subject of human gene thrombopoetin using cDNA ligand murine mpl receptor as a probe. This Enoteca was titrated, and 30 cups of size 150 mm, inoculated with E-Li barreled LE 392 cells (Stratagene Cloning Systems), were infected 410 4 plaque-forming units (PFU). Bowls were incubated overnight at 37C. Were made filtration podyskutowali. These filters were treated with denatured alcohol in a solution containing 1.5 M NaCl and 0.5 M NaOH for 7 minutes at room temperature. These filters were quickly batterbury on filter paper to remove excess solution of denatured alcohol followed by neutralization for 5 minutes in 1 M Tris-Hcl (pH 7.5) and 1.5 M NaCl. Phage DNA was fixed to the filters of the device for cross-linkage (Stratagene Cloning Systems) STATALINKER R with UV energy in 1,200 McAuley. After fixing the filters were washed three times in 0.25SSC, 0.25% SDS and 1 mm EDTA at 65C. After washing, the filters previously hybridisierung in hybridizers solution (5SSC, 5the solution Denhart, 0.2% SDS and 1 mm EDTA), which was filtered through a 0.45 mm filter. Immediately before use) was added processed heated denatured, fragmented DNA salmon sperm (final concentration 100 mg/ml). Filters previously hybridisierung at 65With during the night.

Mouse cDNA TRO full length of pZGmpl-1081 was marked R arbitrary premirovanii using a Labeling System MEGAPRIME DNA tm (Amersham) by the method recommended by izgotovitelej src="https://img.russianpatents.com/chr/215.gif">106pulses per minute, and hybridized overnight at 65C. After hybridization, this hybridization solution was removed, and the filters were propulsives 4 or 5 times in a wash solution containing 0.25SSC, 0.25% SDS and 1 mm EDTA. After propulsive these filters were washed 8 times consecutively at 50With the washing solution. After the last washing, the filters were exhibited before autoradiographically film (XAR-5; Eastman Kodak Co.; Rochester, NY) for 4 days at -70Using the intensifying screen.

The study of autophotography revealed several hundred regions which have hybridisable with labeled probe. Agar plugs were taken in 100 regions for cleaning. Each agar plug during the night were drenched with 1 ml of SM containing 1% chloroform (Maniatis and other Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982). After incubation over night phage from each tube was dissolved in a ratio of 1:1,000 in SN. Volumes that are multiples of 5 ml were inoculated on cells of strain E-Li LE392. Bowl incomelevels overnight at 37With and were prepared lifts for filters, prehybridization, hybridized, washed, and ed is lnyh positive signals from the two main Atlantov and weak signals from other 18. Agar plugs were taken from the positive areas for each of the 20 signals. Agar plugs were processed as described above. Phage alteromonas from each agar plugs were diluted in a ratio of 1:100 in SM and volumes that are multiples of 1 ml, were inoculated with cells of E. coli strain LE392. Bowls were incubated and prepared and hybridisierung lifts phage filters, as described above. Filters were washed at 55With the washing buffer. Autoradiography identified hybridization corresponding to the signal, discrete phage plaques from the three original Atlantov, 8-3-26 10-1-1 and 29-2-1.

Phage isolanti 8-3-2, 10-1-1 and 29-2-1 were identified ZGml-N10 and lambda ZGmpl-H29 respectively. DNA from Atlantov lambda ZGmpl-H8, ZGml-N10 and ZGm1-N were cleaned using phage absorbent LAMBDASORB tm (Promega Corp. Madison, WI) according to the manufacturer's instructions. Insertion of human genomic DNA from phage were separated from the phage vector DNA absorption b I and purified by gel-electrophoresis agarose. All three phage isolante contained sequences that were hybridizations with cDNA probe ligand murine mpl receptor, as shown in the southern blot analysis (Maniatis and others, ibid.). Phage ZGml-H8 of analysisosolution, 2,5, etc., ad 1 and so on, N. a Fragment of 2.5 T. p. N. was subcloned in phagemid BLUESCRIPT II S+ absorbed Xba I (Stratagene Cloning Systems) in order to get plasmogamy pZGmpl-H82.5.

The sequence of the human TPO gene and the encoded amino acid sequence shown in SEQ ID NO:28 and SEQ ID NO:29.

EXAMPLE X. ISOLATION THROMBOPOETIN HUMAN cDNA FULL LENGTH

Human DNA encoding a SRW, full length isolated by the polymerase chain reaction of platelet cDNA of human liver and kidney, using special primers designated by exonic sequences identified on pZGmpl-H82&5 and saved 5’ netransliruemoi sequence of mouse TPO cDNA.

Poly d(T) of the human kidney, liver and lungs, selectively poly (A)+ RNA (Clontech, Palo Alto, CA) was used for synthesizing cDNA of the first barrel. Each reaction was prepared using 4 micrograms of poly(A)+RNA, mixed with 1 mg of oligo d(T)18 (without the 5’ phosphate) primer mPHK (New England Biolab, Beverly, MA) with a final volume of 19 ml of the Mixture was heated to 65C for 5 minutes and cooled on ice. The cDNA synthesis was initiated by addition of 8 ml of 5buffer SUPERSCRIPT tm (GIBCO BRL), 2 ml of 100 MMS., Piscataway, NJ), 2 ml of 1 MCI/ml R-a-dCTP (mrshm, Arlington Heights, IL) and 8 ml of 200 U/ml reverse transcriptase SUPERSCRIPT tm (GIBCO BRL) for each mixture of the RNA primer. Reactions were incubated at 45C for 1 hour and was diluted to 120 ml with TE (10 mm Tris:model HC1, pH 8.0, 1 vV EDTA). cDNA was precipitated twice by adding 50 ml of 8 M ammonium acetate and 160 ml of isopropanol. The precipitate cDNA was transferred again into suspension in 10 ml of TE. The amounts of cDNA first shaft for each reaction were predicted to equal the levels of implementation of 32p-dCTP.

cDNA of the first barrel of mPHK liver, lung and kidney were used in the formulation of the two segments of cDNA, N-end, equal to 1/3, and C-end, equal to 2/3 of the sequence using the polymerase chain reaction. Restricciones website PKK I were introduced into the cDNA segments through a few changes in the Foundation of the genomic sequence using mutagenesis PCR, using primers ZC7422 (SEQ ID NO:20) and ZC7423 (SEQ ID NO:21). The net change of nucleotides generated normal restrictionism Kpnl site without changes in the specified encoding amino acids.

Segment N-end was amplified in 50 ml of reagent containing 5 ng cDNA platelets (in separate reactions for SDATA of deoxynucleotide (Cetus Corp., Emerville, CA), 5 ml of 10the PCR buffer (Promega Cotp., Madison, WI) and 2.5 units of Taq polymerase (Boehringer, Mannheim). Polymerase chain reaction was carried out in 35 cycles (1 minute at 94C, 1 minute at 58C and 1.5 minutes at 72C) followed by incubation for 7 minutes at 72C. Primer Feelings ZC7424 (SEQ ID NO:22) was blocked netransliruemye the region 5’ of the ligand mouse ml and included the ATG initiation codon. Primer of anticosti ZC7422 (SEQ ID NO:20) included the sequence of the region corresponding to axonal 4 and 5 of human genomic DNA TRO.

Segment C-end was amplified in 50 ml of reagent containing 5 ng cDNA platelets (human kidney, liver and lungs, as described above), 80 rmala of oligonucleotides ZC7423 (SEQ ID NO:21) and ZC7421 (SEQ ID NO:23), 5 ml of a 2.5 mm solution of triphosphate deoxy nucleotide (Cetus Corp.), 5 ml of 10the PCR buffer (Promega Corp.) and 2.5 units of Taq polymerase (Boehringer Mannheim). Polymerase chain reaction was carried out in 35 cycles (1 minute at 94C, 1 minute at 65C and 1.5 minutes at 72C) followed by incubation in teceiving, corresponding to axonal 4 and 5 of the human TPO DNA. Primer of anticosti ZC7421 (SEQ ID NO:23) included the sequence of the region corresponding to the non-coding 3’ sequence of the human gene, and included the termination codon of translation.

Amplificatoare products PRC were analyzed by direct sekvenirovanie DNA were subcloned in pGEM-T (Promega Corp.) for further analysis by comparing the sequence of the mouse cDNA and human genomic sequence. The DNA sequence encoding the human TPO shown in SEQ ID NO:18, and the encoded amino acid sequence shown in SEQ ID NO:19. The sequence analysis shows that cleavage of the signal peptide occurs in the amino acid 22 (SEQ ID NO:19), and the Mature protein begins at amino acid 22 (SEQ ID NO:39).

Fragments of human N-endings and endings were cut from pGEM-T, as fragments EcoRI-Kpnl and luigirules in the EcoRI site expressing vector Zem229R. This plasmid was transfectional in KSS 570 cells using Lipofectamine tm (GIBCO BRL). 24 hours after transfection the cultural medium was replaced by fresh medium and cells were incubated for 48 hours in the absence of selective agents. To as described earlier. The results clearly showed that the human TPO in the environment of the cultivation stimulates the proliferation of BaF3 cells expressing the murine MPL receptor.

cDNA made from mRNA in human liver and kidney (obtained from Clontech Laboratories, Inc.) using the reverse transcriptase SUPERSCRIPT tm (GIBCO BRL) in accordance with the specification of the manufacturer. Then got the DNA clones of the human TPO allocated from the liver and kidneys, using 2 PCR reactions (conditions are listed in Table 6). Reactions were carried out in 35 cycles at 94C for 1 minute, 58C for 1 minute. 72C for 1.5 minutes; followed by incubation for 7 minutes at 72°C.

The PCR products were treated with phenol/chloroform/isoamyl alcohol and deposited with I ETON, dried and transferred in suspension in 20 ml of H2O. Then each product into fragments restrictionenzyme enzymes s718 and EcoRI and subjected to electrophoresis on 1% agarose gel. Fragments of length 410, etc., N. (liver or kidney) from reaction 1 and the fragment in 699, etc., N. (liver and kidney) from the Reaction No. 2 were cut from the gel and eleutheropolis by centrifugation of the plates of the gel through nylon wool. PCR product is p-Type, 12301 Parklawn Drive, Rockvilie, MD September 28, 1993 catalogue number 69447), which was cut by EcoRI, which has combined these two products created in the Asp718 site. The resulting plasmid was designated No. 10 (according to cDNA, which is diverted from the kidneys) and No. 28 (containing cDNA designated by the liver).

After sekvenirovaniya DNA were detected unit caused by PCR errors at the 5’ and 3’ from the unique AvrII site in the plasmid No. 28 and No. 10, respectively. To create a DNA TRO fragment of the 5’ EcoRI-AvrII from 826 T. p. N. isolated from No. 10 and a fragment of 3’ from the AvrII-EcoRI was isolated from No. 28. These two fragments were luigirules together with the vector Zem 229R, which was cut by EcoRI. The resulting plasmid was designated pZGmpl-124. This plasmid was deposition in the American Culture Collection Type, 12301 rklawn Drive, Rockville, MD 4 mA year as transformant with catalogue number 69615.

EXAMPLE XI. The cDNA LIBRARY of MEGAKARYOCYTE

For amplification of precursor megakaryocytes in vivo 20 mice were injected into the abdominal cavity 40,000 units of activity (units defined as 500/ml, to obtain half of the maximum speed of cell proliferation F3/MPLR1.1 when the MTT assay (Example VII) recombinant murine thrombopoietin daily (concentrated serum free of condi injection spleen was removed and placed in the CATCH buffer+HEPES balanced salt solution, Henk (HBSS), free from calcium and magnesium, 10 mm Hepes (GIBCO BRL), 1.4 mm adenosine, 2.74 mm teofilina (Sigma Chemical Co., St.Louis, MO) and 0.38% sodium citrate (J. T. Baker Inc., Philipsburg, NJ) with pH adjusted to 7.40 with sodium hydroxide). Five spleens were processed at the same time, they incision, removing the cells between two sieves of stainless steel in a mixture of buffer GATCH+Hepes. After breaking the accumulation of cells in 25 ml th pipette volume was increased to 50 ml, and cells were mixed for 7 minutes in a Sorval centrifuge TJ-6 208g. Each cell sediment was transferred again into suspension in 10 ml of a mixture of CATCH buffer+Hepes and filtered through 130 mm-e nylon sieve to obtain a single cell suspension. The volume was increased to 50 ml due to CATCH buffer+Hepes, and cells were mixed for 15 minutes at 33g. These cells were washed in an additional 50 ml of CATCH+Hepes and were mixed for 10 minutes at 33g. Cellular precipitation was again transferred in suspension in 10 ml of CATCH buffer+Hepes and divided into layers in the three-step Percoll (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) gradient (65, 40 and 27% in 1 x CATCH buffer+Nerey, 12 ml each in 50 ml centrifuge tube) and tsentrifugirovanie for 45 minutes at 833G and was again transferred in suspension in 50 ml of media growth of megakaryocytes (minimum essential alpha-modification of the environment), free ribonucleoside and deoxy of ribonucleoside with 15% inactivated fetal calf serum, 2 mm L-glutamine (media components obtained from JRH Biosciences, Lenexa, KB), 1 mm sodium pyruvate (Irvine Acience, Santa Ana, CA), 1PSN mixture of antibiotics (GIBCO BRL) was added to 1,000 units of the activity of recombinant mouse thrombopoietin/ml (serum-free conditioned medium from KSS 570 cells were stably transactionalist with cDNA mouse thrombopoietin). Then the cells were inoculated in 150 mm plates for culturing tissue in 106mononucleotide cells/ml and were grown in a fully humidified incubator with the content of 6.0% CO2in air at 37C. After three days of growth unattached cells were collected in 50 ml centrifuge tube and cooled on ice. Large cells were inoculated by centrifugation at 33-g for 15 minutes at 4C. Cellular precipitate was again transferred in suspension in 50 ml of CATCH buffer+Hepes at room temperature and were mixed for 10 minutes at 33g. (All Dahl is high purity Mature megakaryocytes. The remaining cells were transferred again into suspension in 15 ml of SATIN buffer+Hepes (volume group) and was displayed on three layers of step gradients fetal bovine syrocki (JRH Biosciences) (65% and 40% of STSN buffer+Hepes) sedimentation (settling) when 1g for 30 minutes. The bottom 5 ml 64% fractions were grouped, it was diluted to 50 ml with a mixture of CATCH buffer+Hepes and was mixed for 10 minutes at 33g. The sediment contained more than 107cells. Cells were studied at acetylcholinesterase by the method of Burstein and others (J. Cell.Physiol.122:159-165, 1985), and it turned out to be Mature megakaryocyte cells with a purity of more than 00%. Then the precipitated cells are lysed (broken) in a solution of thiocynate of Guanica/2-mercaptoethanol to isolate RNA using centrifugation in density gradient of cesium chloride.

cDNA is made from megakaryocytes RNA, as described above in Example VI.

EXAMPLE XII. FLUORESCENCE in situ HYBRIDIZATION MAPPING of the HUMAN TROMBOTICHESKOE GENE

To 1.5 ml tubes of microcentrifuge on ice was added the following: 1 mg ZGml-N8, ZGml-N10 or ZGml-N containing human thrombopoiesis gene, 5 ml of 10 the dATP, 0.5 dGTP, and 0.5 mm dCTP, 5 ml of 5 mm Bio-11-dUTP (5-(N-{N-biotinyl-e-aminocaproyl-3-aminoallyl)-2’-deoxyuridine 5’-triphosphate, Sigma Chemical Co.), 5 ml of 10 mm DTT, 5 ml of DNase has I (1000dilution of 10U/ml stock, Boehringer Mannheim, without RNase), 2.5 ml DNA polymerase I (5 U/ml, Boehringer Mannheim), N2To a final volume of 50 ml After mixing, the reaction was incubated at 15°C for 2 hours in Mikrokreditna Bokela. The reaction was stopped by adding 5 ml of 0.5 M DTA, pH 7.4. Probes were purified using columns for DNA purification Sephadex G-50 (Worthington Biochemical Corporation, Freehold, NJ) according to the manufacturer's instructions. To control the size of the labeled probes in 5-10 ml of each cleaning probe was mixed with 5 ml of gel loading buffer (12.5% ficoll, 0.2% Bromphenol blue, 0.2 M Tris-acetate, 0.1 M sodium acetate, 1 mm EDTA) and then processed 0.7% mingela agarose at a voltage of 80 V, Fragments of lambda Hind III (GIBCO BRL) and fragments PF-Nai III (GTBCO BRL) were used as markers of dimensions base pairs (etc., ad). Centromeric probe, labeled digoxigenin characteristic of chromosome 3 (D3Z1),was obtained from Oncor (Gaithersburg, MD).

Metaphase chromosomes were obtained from cell culture Hell. 100 ml Colcemid (GIRCO BRL, 10 mg/ml) strain were added to the media 10015 mm Petri plate, COI the water was removed and transferred into 15 ml polypropylene conical tube (Blue Max tm, Becton Dickinson). 2 ml of 1PBS (140 mm NaCl, 8 mm KCl, 8 mm Na2HPO4, 1.5 mm KN2RHO4, pH 7.2) was added to a Petri plate for washing with 5 ml sterile plastic pipette, and then it was transferred into a conical tube. 2 ml of trypsin (GIBCO BRL, solution strain) was added to a Petri plate using a sterile 5 ml plastic pipette, and then the Petri plate slightly shaken and placed in an incubator at 37With 3-5 minutes. Then cells were washed with bowl Petri using a 5 ml sterile pipette and added to a tube with environments. Tube for cultivation was centrifugals at 250g for 8 minutes and all but 0.5 ml of the supernatant liquid was removed. The precipitate was again transferred into suspension by tapping, then gently and slowly there was adding 8 ml of 0.075 M KCl (sat preheated to 37C). This suspension was carefully mixed and placed in a water bath at 37With 10 minutes. The solution was centrifugals at 250g for 5 minutes and all except 0.5 ml adosados liquid was removed. The precipitate was transferred into suspension by tapping tarverdian) cells. Thus, in General, was added 8 ml Tube was placed in the refrigerator for 20 minutes, followed by 5 minutes of centrifugation at 250g. The supernatant was usacialis and the process of fixation was repeated two more times. For applying drops of 25 metaphases75 mm pre-cleaned, frozen glass plates (VWR Scientific, Media, PA) on each plate was applied to 5 ml of 50% acetic acid with 20 ml Pipetman tm (Gilson Medical Electonics, Inc., Middleton, WI), and then were transferred to 5 ml of cells in suspension. Plates were left to air dry, and then grew old overnight at 42C in a furnace (oekel Industries, Inc., Philadelphia, PA) before use. The plates were checked for the appropriate growth of metaphases with a microscope equipped with phase contrast condenser. Some preparations of metaphase chromosomes were alight with G-stripe paint Gamesa R66 (BDH Ltd., Dorset, England), photographed and cleaned from paint before use in experiments on hybridization. Drugs on the plates with the rampant human metaphase chromosomes were incubated for 2 hours in 2SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), mystructure buffer Giemsa, pH 6.5 (BDH Ltd) and were filtered before use through a filter of Watman No. 1. Some preparations were first incubated for 45 minutes - 1 hour in an oven at 90With and were left to cool before incubation in SSC. These drugs are then differentiated in a solution of buffer Giemsa, propulsively in H2O and dried in air. The corresponding expansion of the metaphase chromosome is alight with G-band, photographed on a Nikon microscope using film 400 slides kodak Ektachrome tm and pronumerals and stored on the color video system Optronics (Goleta, CA) ZVS-47E CCD RGB and equipment Optimus (BioScan Inc., Edmonds, WA). Drugs cleared of paint for about 20 minutes in 100% EtOh and dried in air before further use. Unused medications plates metaphase chromosomes were stored at -70C.

Hybridization mixtures were prepared in 1.5 ml sterile tubes microcentrifuge, combining 2.5 mg competitive DNA (Cot-1 DNA, GI BRL), 40-60 ng labeled with Biotin phage ZGml-N8, ZGml-N10 or ZGml-N, (containing human thrombopoiesis gene), 7 mg DNA carrier (DNA denatured salmon, Sigma Chemical Co.), 1 ml of 3 M NaAc and 2 volumes of ethanol, the issue is the future 10% dextran sulfate, 2SSC and 50% formamide (EM Science, Gibbstown, NJ). This probe and competitive DNA was dematerialise at 70-80C for 5 minutes, cooled on ice and were pre-annealed at 37C for 1-2 hours. Chromosomes were denaturalise dip each plate in 70% of formamide, 2SSC at 70-80C for 5 minutes, followed by immediate cooling cooled in ice 70% ethanol, then 100% ethanol for 5-10 minutes each. Then these plates were dried in air and heated to 42With just before applying them with 20 ml Gilson Pipetman pipettes hybridizers mixtures. These hybridizers mixture and chromosomes are then covered with a cover glass size 1818 mm, No. 1 (VWR Scientific). Hybridization was performed in a humid chamber overnight at 37C. In some cases, after about 6 hours of hybridization 5-10 ng denatured, labeled digoxigenin D3Z2 centromeric probe (in the hybridization solution, 10% dextran sulfate, 2SSC and 65% formamide) was added to the preparations.

After removal of the SSC at 42C, 35 minutes 2SSC at 42With and 1-3 minutes 4SSC, 0.05% polyoxyethylenesorbitan of monolaurate (Tween-20, Sigma Chemical Co.). After this was pre-incubation for 20 minutes with 4SSC containing 5% skimmed milk powder in a wet chamber (100 ml under cover glass size 2450 mm). For preparations, which included Centroamericano chromosomes 3D3Z1, was conducted by a 45 minute incubation when diluted in the ratio 1:100 labeled with Biotin mouse antidigoxin (Sigma Chemical Co.) 4SSX/5% BSA, then went 3 washing three minutes in 4xSSC, 0.05% Tween-20. We then conducted the stage of paleypalila for all drugs in a 20 minute incubation with saidina labeled with fluorescence (Fluorescein Avidin DCR, Vector Laboratories, Burlingame, CA) (100 ml, 5 mg/ml in 4SSC, 5% skim milk powder) under the cover glass 2450 mm, Then the plates were washed 33 minutes 4SSC, 0.05% Tween-20, then went on a 20 minute incubation biotinilated goat antivenom D (affinity purification,vector Laboratorues) (5 mg/ml in 4">50 mm, the plates were washed again 33 minutes 4SSC, 0.05% Tween 20, and then walked another incubation with Avidya labeled with fluorescent (100 mg/ml in 4SSC, 5% skimmed milk powder) under the cover glass 2450 mm In some cases, the procedure signal amplification was repeated one more time. Final washing was continued for 23 minutes 4SSC, 0.05% Tween-20 and 13 minutes in 1PBS. Plates were installed in protivorechivaya environment, consisting of 9 parts glycerol containing 2% 1,4-diazobicyclo(2,2,2)-octane (DABCO dissolved at 70C) and one part 0.2 M Tris/HCl, pH 7.5, and 0.25-0.5 mg/ml iodide propid. The plate was viewed under a microscope Olympus BH2, equipped with a video of the reflected fluorescent light BH2-RFC, automatic photomicrographic system, color video camera ZVS-47E CCD RGB and red filter FITC/Texas (Brattlebow, VT) production of Congoma Technology Corp. for FITC visualization. Image enlargement metaphase chromosomes were numbered and stored in the camera system video image the human tromboticheskoe gene is distal to the q arm of chromosome 3 in the region 3q26.

EXAMPLE XIII. EXPRESSION CYTOKINES REGION of MOUSE TPO In Sacharomyces cerevisiae

Plasmids pBJ3-5 contains the promoter of S. cerevisiae TR, secretory leader and factor, murine TPO encoding sequence (SEQ ID NO:1) from 237, etc. n 7 to 692, transcriptional terminator TR<2 m sequence for replication in yeast and gene phosphate isomerase triose Schizosaccharomyces pombe (POTI gene for selection in yeast. This plasmid is designed to direct secretion of the protein of mouse TPO containing amino acids 45-196 of SEQ ID NO:2.

To build BJ3-5, pMVRl (Figure 2) were absorbed with SphI and XbaI, and the skeleton of the vector containing the 5’ portion of the promoter TR and terminator TR, was restored. Then, in this frame vector were inserted the following segments:

1) SphI Fragment/Hind III, taken from S114, which contains the 3’ portion of the promoter TR and the leader of the a-factor. Plasmids pBS114 is a yeast Shuttle vector that contains a promoter TR and the leader of the a-factor, after which polylinker sequence, which includes the site of Hind III.

2) HindIII/Sall fragment, generated by PCR, containing the site of Hind III, designed so that it matches the site Hind III, the leader of the a-factor, the site of proteolytic cleavage KEH and sequence of the murine D NO:1, which was given from plasmid pZGmpl 1-1 081 using primers ZC319 (SEQ ID NO: 27) and ZC7318 (SEQ ID NO:26), absorbing with Eco RI and cloning the fragment containing the sequence cytokines region TRO and 5’ non-coding sequence in the website Eco RI from Zem229R {ATCC 69447}. This fragment was changed in the Sall fragment/Xbal cloning it in RSN which was initially absorbed Sall and EcoRI.

The resulting plasmid, designated pBJ3 (Figure 2), then was absorbed BgIII and XhoI to highlight in the free state of complete expression cassette containing a promoter, a leader sequence encoding a TRO and terminator. This BgIII fragment/Xhol were inserted in pRPOT (disclosed in U.S. Patent No. 5,128,321, which is incorporated herein by reference), which was absorbed BamHI and Xhol. The resulting plasmid was designated pBJ3-5.

S. cerevisiae strain JG134 (Mata IgA-52 lu2-A2 RER-A1 atpil:URA3 {fcir about} transformed using pBJ3-5 and pRPOT with treatment lithium acetate (as broadly disclosed in ito and others, J. Bacteriol. 153:163-168, 1983). Transformants were selected by growth on gluconeogenesis environment. JG134/pBJ3-5 and JG134/pRPOT were grown in liquid YEPO environment for three days. The cultural medium was separated from cells by centrifugation and analyzed by examining the proliferation letoaaa as a negative control JG134/pRPOT had no activity. This result indicates that the yeast can identify biologically active form of solid radwaste.

EXAMPLE XIV. The ACTIVITY of RECOMBINANT HUMAN TPO

DNA plasmid from two 5 ml bacterial cultures, cultured overnight, transformed with pZGmpl-124, privatelives using lysis of cells alkaline, after which DNA was contacted with the resin at a high salt content (using a set of sampler Magic Minipreps from tm Propega Corp.). DNA was eleutheropolis with 75 ml of 10 mm Tris, 1 mm EDTA, pH 8.0.

Cell culture KSS 570 under 50,000 cell/well were transfection with DNA pZGmpl-124. 20 ml when diluted LIPOFECTAMINE tm (GIBCO BRL) in a ratio of 1:10 was added to 20 ml of DNA plasmid and 160 ml of serum-free media (environment F/DV {mixture in a ratio of 1:1 DMEM and F12 Us} adding 10 mg/ml of fetuin, 2 ng/ml selenium, 5 mg/ml insulin, 10 mg/ml transferrin, 2 mm L-glutamine, 110 mg/ml sodium pyruvate, 25 mm HEPES and 0.1 mm solution of non-essential amino acids (GIBCO BRL) for 30 minutes at room temperature before adding to the cells KSS 570 and incubation for 4 hours at 37C. 200 ml of Growth Environments (DMEM (Biowhittaker), supplemented with 2 mm L-glutamine, 110 mg/ml sodium pyruvate, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 0.01 mg/ml neomycin, 25 mm HEPES, 10 m was replaced with Growth Medium, containing 5% fetal calf serum and were incubated at 37C for 4 hours.

The air-conditioned environment of the transfectants KSS 570 then were tested for ability to induce cell proliferation in BaF3 cells expressing the murine MPL receptor. These cells were grown in media BaF3 (RPMI 1640 medium (JRH Biosciences) supplemented with 10% fetal calf serum, 2 mm L-glutamine, 1 mm sodium pyruvate, 10 mm HEPES, 57 mm beta-Mercaptoethanol, 0.5 mg/ml penicillin, 0.5 mg/ml of streptomycin, 0.1 mg/ml neomycin and 4% V/V conditioned medium from cultures of cells WEHI-3 (murine interleukin-3, in addition to culture. Produced jointly by Biomedical Products)). To research cells F3 were diluted and re-translated in suspension in the medium F3 free of IL-3, up to 10,000 cells/100 ml of conditioned medium from cells KSS 570, transfection pZGmpl-124, and these cultures were incubated at 37C. Then, these cells were studied Mizulina on the elongation of the cells after 30 minutes and after 24 hours. Negative controls, consisting of environment F3 without IL-3, and a positive control conditioned medium from cells KSS 570, transfection with DNA of mouse TPO, was also investigated. , the number of movements of the cells were positive control and a considerable elongation of the cells was observed in cells transfection pZGmpl-124.

EXAMPLE XV. AFFINITY PRECIPITATION of RECEPTORS

150 mm bowl for culturing tissues containing cells producing TRO or normal KSS cells were labeled for 18 hours, 10 ml of MEM, Dulbecco without methoinine containing 2 mm L-glutamine, antibiotics and 200 MCI of 35S-Express (Amersham, Arlington Heghts, IL).

After incubation over night used the medium was collected and concentrated 15-fold using Centriprep concentrator-10tm (Amicon, Inc.). Received 0.7 ml of concentrated supernatant was mixed with 40 ml of soluble MPL receptor with the end in the form of a poly-histidine, which was linked to CNBr-Sepharose 4B (Pharmacia) that was specified by the provider. The mixture was incubated for 2 hours on ice with shaking.

Cells were washed once with PBS, and then were destroyed in 1 ml of RIP buffer A (10 mm Tris, pH 7.4, 1% of deoxycholate, 1% Triton X-100, 0.1% SDS, 5 mm EDTA, 0.15 M NaCk). Lysates were tsentrifugirovanie to remove insoluble substances, and added 10 ml MPL-Sepharose, as previously indicated.

Then MPL-Sepharose was deposited at low speed centrifuge, and used the environment and the supernatant of cellular Liz is allelse 40 ml of 2a test buffer (10% glycerol, 4% SDS, 50 mm Tris, pH 7.0, 1 mm EDTA, 0.05% Bromphenol blue) containing 4% beta-mercaptoethanol.

The samples were boiled for 5 minutes, and 18 ml was loaded on the ini-gel with 10-20% gradient (Integrated Separation System), then subjected to electrophoresis at 100 V for approximately 2 hours. The gel was fixed for 30 minutes (40% methanol, 16% glacial acetic acid in distilled water), then otshatyvalsya to AMPLIFY tm (Amersham) for 20 minutes. After drying, the gel was exposed before the film during the night. Band -70 KD was very noticeable in the interval corresponding to the used media from cells transfection cDNA TRO. This band is not present in the media from cells KSS or lysates of cells from each cell line.

From the foregoing it can be understood that although specific examples of the invention described herein for purposes of illustration, various modifications can be performed, without departing from the essence and spirit of this invention. Accordingly, this invention is not limited by anything except the attached claims.

Claims

1. Selected polynucleotide containing fragments is in store protein, which stimulates MPL-dependent proliferation or differentiation of the myeloid or lymphoid precursors.

2. Selected polynucleotide under item 1, where this protein is a protein of the mouse.

3. Selected polynucleotide under item 1, where this protein has the amino acid sequence of SEQ ID NO:2 from amino acid residue 45 to amino acid residue 206.

4. Selected polynucleotide, which is characterized by a nucleotide sequence in accordance with the genetic code determines the amino acid sequence at least 80% identical to SEQ ID NO:2 from amino acid residue 45 to amino acid residue 206, and encodes a protein that stimulates MPL-dependent proliferation or differentiation of the myeloid or lymphoid cells.

5. Selected polynucleotide selected from the group consisting of (a) DNA fragment containing the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 237 to nucleotide 722; (b) polynucleotide encoding a protein characterized by the amino acid sequence of SEQ ID NO:2 from amino acid residue 45 to amino acid residue 206; (c) allelic variants of (b); and (d) polynucleotide, essentially corresponding to the DNA fragment, the 722, moreover, the specified polynucleotide encodes a protein that stimulates MPL-dependent proliferation or differentiation of the myeloid or lymphoid precursors.

6. Selected polynucleotide, essentially corresponding to the DNA fragment selected from the group consisting of (a) inserting Eco RI-Xho I in plasmid pZGmpl 1081-a(ATCC 69566); (b) DNA fragment selected polynucleotide that at least 80% identical to the nucleotide sequence of a), and the specified polynucleotide encodes a protein that stimulates MPL-dependent proliferation of myeloid or lymphoid precursors.

7. The recombinant vector containing the regulatory elements necessary for expression of foreign DNA sequences in eukaryotic cells, and the DNA fragment that is functionally associated with the specified elements and containing a nucleotide sequence selected polynucleotide under item 5, and ensuring the expression of the indicated fragment of DNA in eukaryotic host cells.

8. The recombinant expression vector according to p. 7, further containing a secretory signal sequence, is functionally associated with the DNA fragment.

9. A method of obtaining a culture of cells of microorganisms, expressyour expression under item 7 and selection of cells, synthesizing the specified hematopoietic protein.

10. A method of obtaining a culture of cells under item 9, where the specified cell is a fungal cell.

11. A method of obtaining a culture of cells under item 9, where the specified cell is a yeast cell.

12. A method of obtaining a culture of mammalian cells expressing the hematopoietic protein, including preparation of mammalian cells, introduction of recombinant expression vector under item 7 and selection of cells synthesizing specified hematopoietic protein.

13. The method of obtaining hematopoietic protein, providing for the cultivation of cells obtained by the method according to p. 9 or 12, and removing the hematopoietic protein from the culture medium.

Priority points

14.02.1994 on PP.1-13.

 

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