Recombinant tissue plasminogen activator and method thereof

 

(57) Abstract:

The invention relates to the production of tissue plasminogen activator (enhanced APT) with prolonged biological half-life existence, increased resistance to heat and acids and effective as an inhibitor of inflammation around the site where the formed clot. To obtain hold culturing host cells transformed by a recombinant DNA containing a specific DNA sequence encoding the analogue tp. 2 C. p. F.-ly, 3 tables. 29 Il.

The invention relates to a new improved active tissue plasminogen (advanced APT), which has a prolonged half-life existence in the body and increased stability to heat and acids, which can be used to suppress inflammation around the area of blood clots.

The invention also relates to a method get called tissue plasminogen activator using recombinant DNA technology and tools used for its implementation.

It is known that the human tissue plasminogen activator (PLA) has useful fibrinogen in the phase of circulation in the body it activates not as effective as conventional thrombolytic means, streptokinase (SK) and urokinase (UK). Known amino acid sequence of human APT and nucleotide sequence of cDNA encoding human APT (Pennica. D., et al., Nature, 301, 214-221, 1983). It is also known that human APT dissolve clots venous and arterial blood. In large-scale clinical studies have indicated that human APT, administered intravenously, is effective in re-perfusion zakuporivaniya coronary artery in a patient with acute myocardial infarction.

However, a disadvantage of the use of this drug in the treatment of diseases associated with thrombosis, is extremely short half-period of the existence of its enzymatic activity in the blood (Rijken, D. C., et al., Thromb. Heamost. 54 (1), 61, 1985, Hubert, E. F., et al., Blood, 65, 539, 1985). When used for the treatment of human APT have to apply as a continuous intravenous injection with a high dose.

It is known that naturally occurring human APT has a domain structure, starting from the N-Terminus of the molecule followed by Fingermen, domain FRE (growth factor of the epidermis), two domain "Kringle 1 and Kringle 2 and domer serine protease. In the work Rijken et al., notes (Rijken, D. C., et al., Thromb. Heamost., 54 (1), 61, 1985), what neprodolzhitelnogo APT, in addition to domain serine protease. In the work Zonneveld et al. (Zonneveld, A. J. V., et al, Proc. Natl. Acad. USA., 83, 4670, 1986) also notes that fingerman, domain FRE domain "Kringle 2" can be important for fibrinolitikami activity of naturally occurring human APT, as well as to maintain fibrisolvens activation APT. However, so far not developed any specific measures to maintain fabriksgasse activity possessed by naturally occurring human APT, and his fibrisolvens activity, as well as for prolonging the biological half-time of existence.

In the published lined the patent application of Japan N 48378/1987 describes the PLA obtained by deletion 87-175 amino acids found in human nature APT, in which "Kringle 1" deleterows. This APT different additional induced point mutation in the region of the growth factor of the epidermis. In the patent application of Japan revealed that the modified PLA has the ability to bind to fibrin, but the interaction with the inhibitor of tissue plasminogen activator is weakened.

In Europatent N 241208 describes the PLA obtained by deletion 92-179 amino acids found in nature criticisme activity.

In addition, Europatent N 231624 discloses a modified APT with a prolonged half-life existence. Modified APT with F-EGFK2-A - sequence, devoid, domain "Kringle 1", however, whatever the specific method thereof is not shown. In light of the above cited it is clear that the modified PLA in accordance with the invention must be different from that found in nature APT amino acid sequence in the internal domains.

As a result of extensive research, the applicant received an improved APT, which contains finger domain FRE-domain, the Kringle 2 domain and the domain serine protease, but the first Kringle 1" domain deleterows in the specific site, and the site linking domains "of the Kringle 2 and the serine protease introduced mutation, resulting in enhanced APT exhibiting excellent resistance to heat and acids, with markedly prolonged biological half-life existence and pronounced anti-inflammatory activity and at the same time preserving the desirable properties of naturally occurring human APT.

The invention relates to an improved APT. APT ACC is mini APT and shows the best properties.

Enhanced APT in accordance with the invention is a polypeptide having the amino acid sequence represented by the General formula shown in Fig.28-29, where R is a direct bond, Y represents A-Ile-B (A denotes Arg or Glu and B represents lys or Ile), preferably Glu-Jle-Lys. H2N denotes aminocore and-COOH indicates carboxysomes).

In the invention, the term "enhanced APT" is used to refer to similar APT, in which A and B represent the following amino acids respectively:

enhanced APT (II): Arg, Lys;

enhanced APT (V): Arg, Ile;

enhanced APT (VI): Glu, Lys;

enhanced APT (VIII): Glu, Ile.

The invention is also directed to the expression proposed similar APT using the techniques of recombinant DNA. This is related to the new DNA encoding enhanced APT, and vectors the expression of recombinant DNA.

In Fig.1, 2 shows a sequence of 16 oligodeoxynucleotides used to construct the synthetic fragment of the gene encoding enhanced APT (II) of Fig.3 - 4 - slice synthetic gene for constructing usovershenstvovannye 16 oligodeoxynucleotides, it is shown in Fig.1 - 2 of Fig.5 - the method of constructing an improved APT (II) (figure black area, shaded area and white area denotes the region, encoding respectively the Mature protein of the PLA, the region encoding preprepared and noncoding region of Fig.6 is a method of checking a fragment of the synthetic gene block IV by determining the sequence of DNA bases by dimethoxymethane and method 7-DEAZA; Fig.7 - the method of constructing an expression vector pVY1 in animal cells and integration of DNA enhanced APT in pVY1; Fig.8 - 13 DNA sequence encoding enhanced APT (II) and enhanced APT (V); Fig.14 - 19 - amino acid sequence derived from the DNA sequences encoding the enhanced APT (II) and enhanced APT (V); Fig.20 - restriction enzymes and functional map of plasmid pTPA 2, with a fragment of the Eco R1-Xho (about 1000 base pairs) of the natural gene APT, integrated into the vector pBR322 at the sites of cleavage Eco R1 and Bam H1; Fig.21 - mp9 (advanced APT (II) with the fragment BgL11-Xho 11 (about 1500 base pairs) of the gene, enhanced APT (II) integrated into double-stranded DNA of M13 mp9 in the website of rassal nature of the PLA method S-2251 in the presence (+Fb) and absence (-Fb) Deputy of fibrin; in Fig.23 - the change in the activity of advanced APT (VI) and native APT in the blood of the rabbit over time; Fig.24 - change of residual activity of advanced APT (VI) after heat treatment; Fig. 25 - inhibition enhanced APT (VI) factor, activating lymphocytes (LAF); Fig.26 - activated using denatured protein, enhanced APT (VI) of Fig.27 - degradation of denatured protein under the action of an improved APT (VI).

Below details the method of obtaining recombinant DNA and transformed cells.

A method of obtaining enhanced APT.

The gene encoding the natural APT, on the basis of which to obtain APT the present invention, separated from the Bank cDNA made from cells of human Bowes melanoma. Poly A+RNA extracted from cells of human Bowes melanoma and fractionary by centrifugation in a density gradient of sucrose. Then take a small amount of fractionated poly (A)+RNA and the fraction of mRNA encoding a gene APT identify method detribalization using oligonucleotide probe capable of recognizing a specific sequence of an mRNA up the up the screening with a probe to identify mRNA APT, described above. Because not selected a single clone containing the complete gene sequence of the PLA, the missing sequence of bases synthesize DNA synthesizer with obtaining the desired gene. Then the desired gene construct by induction a site-specific mutations.

Fragment Eco R1-Xho 11 occurs in nature gene APT (about 1000 base pairs), part of which demeterova N = end, is introduced into the vector pBR332 in the sites of cleavage Eco R1 and BamH1 data pTPA2. Strain (E. coli HB 101/pTPA2) obtained by transformation of E. coli with this plasmid deposited with Institute for fermentation research Agency of industrial science and technology of Japan under the registration number P-9649 (FERM BP-2107). Restriction and functional map of plasmid pTPA2 shown in Fig.20.

Gene enhanced APT inserted into the plasmid pVY1.

Plasmid pVY1 get legirovaniem BamH1 fragment-Kpn1 (about 2900 base pairs) plasmids pRSV10 (manufactured by Pharmacia fine, Chemicala) with a fragment from cleavage Eco R1 plasmids pAdD26SV (A) N 3 (N) (obtained from Dr. Hiroshi handa from the University of Tokyo (after receiving both blunt ends. Accordingly, this vector contains the cDNA of the gene dihydrofolate-reductase we are the site of insertion of the gene enhanced APT and intron, and a polyadenylation sequence, below the gene. The gene of the present invention may be embedded in another suitable expression vector. The expression vector being introduced later in suitable host cells with obtaining transformants. As the host cells can be used prokaryotic cells, as well as E. coli, Bacillus subtilis, etc., eukaryotic microorganisms such as yeast, etc., and cells of higher animals. As a representative of E. coli are commonly used strain JM109 strain W3110, Q, etc. belonging to the strain K12, as a representative of the use of Bacillus subtilis strain BD170, strain BR151, etc. From yeast, you can use strain RH218, strain SHY1, etc. of the yeast Saccharomyces cerevisiae.

The expression is usually used plasmid vector or phage vector containing the replicon, derived from species compatible with the host cell, and a regulatory sequence. Examples of vectors for E. coli are, for example, plasmid pBR322, pUC18, pUC19, and so on, a phage, such as qt , Charon 4A, and so on, M13 phage, etc. as vectors for Bacillus subtilis can be used pUB110, pSA2100 and so on, and as a vector for yeast, you can use YRp7, YEp61 etc.

The vector must bear a promoter that can Express the desired protein. As the promoter is and you can use cultured cells of animals, such as kidney cells rhesus monkeys, cells of larvae of the mosquito, kidney cells of the African green monkey, mouse fetal fibroblast cells of the ovary of the Chinese hamster, human fetal kidney cells, tissue cells, the eggs of butterflies, human cervical epithelium-like cells, human myeloma cells, mouse fibroblasts, and so forth. As a vector, you can use early promoter, SV40 late promoter, SV40, SV40, carrying the promoter from eukaryotic gene (for example, estrogen-induced gene of avian ovalbumin gene interferon, glucocorticoid-induced gene tyrosine aminotransferase gene thymidine kinase, early and late genes of adenovirus, gene phosphoglycerate-kinase, the gene-factor, and so on), the virus bovine papilloma or derived from these vectors.

In addition, it is known that the PLA, secreted and produced by cells that have different N-end depending on differences in the sites of cleavage.

In the case of secretion and production of the PLA using culture cells as a host, the method of splitting the signal peptidase or protease varies depending on the type of cells, so that you can get and APT having different N-end.

To transform a host using an expression vector with an integrated genome enhanced APT in the case of E. coli, you can use the method of Hanahan, Hanahan, D. J. Mol. Biol., 166, 557, 1983), in the case of manipulation of animal cells can be used calcium phosphate method (Vander Eb, A. J. and Graham, F. L., Method in Enrymoloqy, 65, 826, 1980, Academic Press) and so on.

As described above, enhanced APT is suitable for the treatment of various acquired diseases, including vascular coagulation (even deep veins), pulmonary embolism, peripheral arterial thrombosis, embolism due to damage to the heart or peripheral arteries, acute myocardial infarction and thrombotic attack.

As found in human nature APT, the advanced APT is particularly suitable for the treatment of acute myocardial infarction. As recently proven, naturally occurring human APT effective for dissolution of obstructive coronary artery thrombus, regeneration of myocardial perfusion and restore 70 mg for 1-3 hours. Enhanced APT differs prolonged biological half-time of existence in the blood and therefore effective in cases that found in human nature APT. It is expected that improved the PLA may provide clinical effect similar to natural human APT, at a dose of about 10% of the dose that is recommended when using naturally occurring human APT, even after a single dose.

In addition, the advanced APT the present invention exhibit the following valuable properties, which still were not known in relation to native human PLA and modified APT.

a) anti-Inflammatory activity.

At the site of thrombus detected not only the formation of the thrombus, but also the formation of degradation products of fibrin or trace amounts cinena. It is known that these substances are inducing inflammation activity and cause, therefore, inflammation in the area of the clot. For this reason, it is desirable that the tool used for the treatment of thrombosis, possessed not only thrombolytic activity, but also anti-inflammatory activity.

The result is that the conduct is the basis of two functions.

One of them is that enhanced APT inhibits the biological activity of interleukin 1 (IL-1), which is one of the mediators of the inflammatory reaction. IL-1, produced by macrophages, as it participates in the inflammatory response by hyperthermia, accelerate growth of the fibroblast, the production of collagenase in synovial cell membrane, and so forth, or by accelerating the synthesis of prostacyclin in vascular endothelial cells. It is also known that IL-1 acts on liver cells, accelerating the production of proteins (serum amyloid protein, fibrinogen, and so on) in the acute phase, which increases inflammation. The applicant has found that an improved APT inhibits the activity (LAF activity) to increase the mitogenic reactivity of mouse thymocyte, which is one of the biological activities of IL-1.

Another function is that enhanced APT has an affinity for denaturirovannogo protein (denaturirovannogo immunoglobulin G, denaturirovannogo albumin, and so on), resulting from inflammation at the site of thrombus, and additionally has the property to be activated under the effect of this is the denatured protein in the area of inflammation, and inflammation may be temporarily weakened. The applicant confirmed by gel electrophoresis in dodecyl-sulfate sodium, which improved the PLA decomposes only the denatured protein.

As shown in Fig. 26, activation and selectivity enhanced APT under the action of denatured protein are obvious. With immunoglobulin G, treated with HCl, with smaller concentrations, shows the same activity, and fibrinogen treated Enrichment. On the other hand, normal immunoglobulin C shows the activating steps towards improved APT even at concentrations equal to 500 mcg/ml.

Preventing re-occlusion after restoring perfusion clogged blood vessel.

It is known that in the treatment of thrombosis natural APT celebrated the re-occlusion with high frequency after restoration of blood flow is occluded blood vessel. For this reason, exercise combination therapy with inhibitors of coagulation platelet or anticoagulant. However, combination therapy encompasses problems drug interaction, control of dosages, such efficiency is occlusion.

Advanced APT the present invention has the ability to prevent re-occlusion due to two types of activity.

The first type is the prevention of a rapid decrease in the concentration of the PLA after the introduction of an improved APT due to the prolonged duration of action, which leads to the elimination of the symptom Stewart-Holmes and thereby prevents the occurrence of re-occlusion.

The second type is that by preventing damage to vascular endothelial cells induced IL-1-mediated inhibited coagulation of platelets, which prevents the occurrence of re-occlusion.

C) Increased resistance.

Protein drugs, as a rule, unstable, therefore, it is desirable to store the drugs in the frozen dry conditions or at low temperatures in solution. With the introduction of the plasminogen activator to a patient with acute myocardial infarction there is a need to carry out the procedure within a few hours after the start of the attack in order to reduce the mortality rate. In this case, the desired stable medication that can groovebot, handling acids, etc. during the preparation. In particular, in relation to advanced APT the present invention, which is produced by cell culture, it becomes possible to remove retrovirus cell origin, which is known to be unstable to heat.

Below the invention is described more specifically with reference to examples, however it is not restricted by them. Unless something else, recombinant DNA produced in accordance with the laboratory manual.

Maniatis T, etc., Molecular cloning: a laboratory manual, Cold spring Harbor Lab, Cold spring Harbor, new York (1982).

Example 1. Cloning the DNA of the PLA.

Cells of the human Bowes melanoma (purchased from Dr. Roblin, R. National Institute for cancer research, USA) were cultured according to the method Opdenakker et al. (Opdenakker, G., et al., Eur. J. Biochem, 131, 481-487 (1983)). The purpose of induction of mRNA of the PLA into the culture mixture was added TN (12-O-tetradecanoylphorbol-13-acetate) at a final concentration of 100 ng/ml and then cultured for 16 hours. Then total cellular RNA extracted from cultured cells in line is once with oligo-dT cellulose (manufactured by Pharmacia fine chemicals) poly (A)+RNA is separated from whole cell RNA. As a result of number of approximately 10ocells receive about 400 μg of poly(A)+The RNA.

This poly(A)+RNA fractionary by centrifugation in a density gradient of sucrose in the traditional way. Select the part of fractionated poly(A)+RNA, and spend the dot-blotthybridization (Perbal, B., Apractical Gube to Molecular Cloninq, 410, 1984, John Wiley and Sons, Inc) using an oligonucleotide probe specific for the mRNA of the PLA. Probe (probe Y) used in this case has a sequence of bases 5'-GCNNGGCAAAGATGGCA-3', which is complementary region of the mRNA that encodes amino acid residues from +291 to +297 in the sequence of the PLA described Pennicaetal, and synthesize-cyanophosphonate method using a DNA synthesizer, model A, (produced by Applied Biosystems). Synthesis of a DNA oligomer, cleavage of the protective group cleavage from the resin and purification is carried out in accordance with the manual DNA synthesizer, Model A. The radioactive isotope tagging probe Y at the 5'-end is carried out in accordance with laboratory manual, using T4-polynucleotide-kinase (manufactured Taka-RA Shuso Co., Ltd.) t and(32P) ATP.

Probe Y strongly hybridization 10 μg of poly(A)+RNA from fractions M; 3 μg double-stranded cDNA is synthesized using reverse transcriptase (manufactured Biochemical industry Co., Co., Ltd.) in accordance with the method after Gubler-Hoffman (after Gubler, U. and Hoffman, B. J., Gene 25, 263, 1983), and add to double-stranded cDNA at the 3'-end deoxy C-chain in accordance with the method Denq-Wu (Denq, G. R. and Wu, R., Nucleic Acids Res., 9, 4173, 1981). Then double-stranded cDNA, elongated deoxy C-chain is subjected to gel filtration on sepharose CL 4B (manufactured pharmaceutical fine chemicals) to remove low molecular weight nucleic acids having less than 500 base pairs. After this cDNA is subjected to annealing using pBR322 (manufactured, Bethesda, Maryland research), containing deoxy G-chain in the website Pst1, using conventional methodology. The mixture obtained after annealing transform competent cells of E. coli HB101 (manufactured by Takara Shuso Co., Co., Ltd.). As a result, the Bank cDNA, consisting of approximately 4,000 independent transformants.

This cDNA is subjected to hybridization of colonies using a probe Y described above, in accordance with the method of Woods (Woods, D., Focus, 6 (3), 1, 1984), manufactured Bethesda research lab.), getting clones interacting with the probe Y. Among clones reveal pTPA1 clone containing the most glennwood 7-DEAZA (S. Mizusawa , et al., Nucleis Acids Res., 14, 1319, 1986). The result revealed that the plasmid pTPA1 contains a sequence of bases from Ty+441to Ay+2544for gene APT, described Pennicaetal.

Example 2. Designing advanced APT (II).

Plasmid pTPA1 shown in example 1, the N-terminal region is sufficient to build advanced APT (II) who is devoid of the Kringle 1 domain. Therefore, insufficient DNA segment synthesized as described above using the DNA synthesizer A (manufactured by Applied Biosystems). The sequence of bases of the synthesized oligomer and full synthesized by the sequence shown in Fig. 1-4. The specific method of designing improved APT (II) with the use of these oligomers is shown in Fig. 5-6.

2-1). Design unit IV (fragment Bql II-Eco R1, about 480 base pairs).

The fragment block IV in Fig. 5 was obtained as follows.

First, in accordance with the laboratory management, 40 pmol each of the synthetic oligonucleotides 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, shown in Fig. 1-2, phosphorylate 10 units of T4-polynucleotide-kinase (manufactured by Takara Shuso Co., Co., Ltd.) at a temperature of 37t phenol. After precipitation by ethanol precipitation, dried under reduced pressure and dissolved in sterile distilled water. After settling 40 pmol of each oligomer in 150 µl of a solution containing 6 mm Tris-HCl (pH 7.5), 20 mm NaCl, 7 mm MgCl2and 0.1 mm EDTA at a temperature of 80oC for 5 minutes, at a temperature of 60oC for 5 minutes and at room temperature for one hour, in the respective blocks of the block I (oligomers 1, 2, 3 and 4), block II (oligomers 5, 6, 7, 8, 9 and 10) and block III (oligomers 11, 12, 13, 14, 15 and 16 by precipitation with ethanol and drying under reduced pressure. The residue is dissolved in 40 ál of sterile distilled water. The reaction is carried out in 400 µl of the reaction solution at a temperature of 4oC for 15 hours, using the kit for DNA ligation (manufactured by Takara Shuso Co., Co., Ltd.). After precipitation with ethanol and drying under reduced pressure the residue is dissolved with terilee distilled water: in case of unit I (1) carry out gelelectrophoresis in 5% polyacrylamide (laboratory manual), is separated and purified using conventional methods (laboratory manual), a fragment of about 100 base pairs, and in the case of block II (2) and block III (3) gel electrophoresis is carried out in a 3% agarose gel (the g) fragments of about 190 base pairs.

Then 0.1 mg, 0.2 µ g and 0.2 µg of fragments of unit I, unit II and unit III, respectively, are ligated using the above kit for DNA ligation. Perform gel electrophoresis at a concentration of agarose 1.5 % in order to allocate fragment Bgl II-Eco R1 (block IV) size of about 480 base pairs. Then DNA isolated from agarose gel using electroelution. This DNA is then fosfauriliruet in 100 μl of reaction solution at a temperature of 37oC for one hour using 10 units above T4-polynucleotide-kinase, and then treated with phenol, precipitated with ethanol and dried under reduced pressure.

This synthetic fragment of the gene and the sequence of bases of unit IV is confirmed, determining the sequence of bases in accordance with dimethoxymethane using a phage vector M13. Specific methods shown in Fig. 6. After ligating the above fragment Bgl II-Eco R1 block IV with M13 mp18 DNA (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.), cleaved with restriction enzymes BamH1 (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.) and Eco R1 (manufactured by Boehringer Mannheim-Yamanouchi Co. Co., Ltd. ) determine the sequence of its bases with a set of sequencing M13 (made Taraka Shuso Cnym enzyme Bgl11 and the site of cleavage with restriction enzymes BamH1 are ligated isodisomy location through (BamH1 - split end Bgl11-site cleavage), and legirovannye fragment can be split with restriction enzymes Xho 11, resulting in a natural ends splitting Bgl 11 and Bamh1, respectively.

For a more accurate determination of base sequence of the phage M 13mp18 (including the fragment block IV) infect a strain of E. cjli JM109 in accordance with the method of Messing/J. Messing, Methods in Enzymology, 101, 20-78 (1983)), and then get double-stranded DNA (replicative). After splitting the DNA (50 µg) restriction enzymes Xho 11 (manufactured by Boehringer Mannheim-Yamanouchi Co) and Eco R1 carry out gel electrophoresis in 1.5% agarose gel slice (block IV) about 480 base pairs. This DNA is extracted by Electrosila. After ligation of extracted DNA with M13mp19 DNA (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd) cleaved with restriction enzymes Eco R1 and BamH1 similar way, using the kit for DNA ligation, determine the sequence of bases. As described above, this sequence can be checked more accurately determining the sequence of both DNA using M13mP18 and M13mp19. In addition, double-stranded replicative DNA M13mp19 (unit IV) are obtained using the above method. After the times, while highlighting fragment (block IV) size of about 480 base pairs.

2-2). The selection unit V (fragment Eco R1-Bal1, about 1250 base pairs).

From clone pTRA1 obtained in example 1, isolated plasmid DNA in large quantities in accordance with the method described in the lab manual, as shown in Fig. 5. After splitting 70 μg of this DNA restriction enzymes Bal1 (manufactured by Takara Shuso To. , Ltd.) and Nar1 (manufactured by Niro Gene Co., Co., Ltd.) perform electrophoresis with 0.8% agarose gel, while highlighting fragment Nar1-Bal1 (about 1540 base pairs). DNA are elektrobudowa. After a further partial digestion of this DNA with restriction enzymes Eco R1 carry out electrophoresis and a 0.7% agarose gel, releasing a fragment of Eco R1-Bal1 (about 1250 base pairs). DNA are elektrobudowa.

2-3). Constructing gene enhanced APT (II) of unit IV and unit V

As shown in Fig. 5, the gene enhanced APT gets as follows.

After doping unit IV (fragment Bgl11-Eco R1, about 480 base pairs) obtained in example 2-1, block V (fragment Eco R1-Bal1, about 1250 base pairs) obtained in example 2-2 using the set for igenom pressure precipitate digested with restriction enzymes Xho 11 in the traditional way. Then carry out electrophoresis in 0.8% of agarose gel in order to select a fragment Bgl 11-Xho 11 (about 1500 base pairs, contains a gene enhanced APT). Then elektrobudowa produce DNA. The full sequence of the bases thus obtained gene enhanced APT (II), shown in Fig. 8-13. Deduced amino acid sequence is also shown in Fig. 14-19.

Example 3. Constructing gene enhanced APT V, VI and VIII.

Constructing gene enhanced APT V, VI or VIII, carried out on the basis of the gene enhanced APT (II) with reference to the following publications.

Genetic conversion carried out by the method of induction of site-specific mutations.

Publications: Zoller, M. J. and Smith.M., Method in fermentology, 100, pp. 468-500 (1983), Zoller, M. J. and Smith. M, DNA, 3, pp. 479-488 (1984), Morinaga Th. and other Biotechnology p. 636-630 (July 1984), J. Adelman. P. and others , DNA, 2, pp. 183-193 (1983), 6. Manual sequencing of M13 (Fig) published gene Saenz room Co., Co., Ltd.).

3-1). Constructing gene enhanced APT (V).

A) the Creation of M13mp19 (APT/P/) for mutation engine.

A fragment of the gene enhanced APT (II) described young what fatty (manufactured by Takara Shuso Co., Ltd.). The product of ligation transferout competent cells E. cjli JM109 (manufactured by Takara Shuso Co., Co., Ltd.). Each clone, giving colorless sterile spot, used to infect E. Coli JM109. Single-stranded DNA allocate ITZ culture supernatant, and double-stranded (replicative DNA extracted from cells of E. cjli in accordance with the method of messing (messing, J. methods in fermentology, 101, pp. 20-78, 1983). When analyzing the nature of these double-stranded DNA after cleavage with restriction enzymes Pst1 using agarose gel electrophoresis get the clone mp9 (enhanced APT (II), in which the gene of the PLA (II) insertion in DNA mp9 in the desired direction, as shown in Fig. 21.

After cleavage of these DNA by restriction enzyme Pst carry out electrophoresis with 0.8% agarose gel, where the clone mp9 (enhanced APT (II) shows a simple strip in position 7300 base pairs, 840 base pairs, 430 base pairs and 80 base pairs, approximately. Single-stranded DNA from this clone used in the subsequent experiments on the induction of site-specific mutations.

B) Synthesis of primers capable of inducing site-specific mutation.

Synthetic oligonucleotide ispolnitelnitsam method using a DNA synthesizer, model 380 A (manufactured by Applied Biosystems). Synthesis of a DNA oligomer, removing the protective group cleavage from the resin and purification is carried out in accordance with the instruction manual DNA synthesizer 380 A. To induce mutations in specific customers receive primer (1), able to induce site-specific mutation and primer (2) for sequencing by dimethoxymethane using phage vector M13 (J. Carlson. and others, journal of biotechnology, 1, page 253, 1984).

Shows amino acid and nucleotide sequences for enhanced APT (II). Primer (1) capable of inducing mutation differs tart base from gene sequence enhanced APT (II) (see tab.1).

C) Inducing site-specific mutations.

Below is a way to create a clone containing the sequence of bases in the primer (1), is able to give mutation, namely gene enhanced APT (IV). After annealing (renaturation) single-stranded DNA as described in example 3,3-1), A) clone mp9 (advanced APT (II)and primer (1) product renaturation converted into double-stranded DNA, which then transform E. coli JM109. Then, using a primer for sekvenirovaniya APT (II), namely gene enhanced APT (V). From this clone extract double-stranded (replicative) phage DNA and produce gene enhanced APT (V).

the 5'-end phosphorylation of synthetic oligomer.

DNA primers (1) to induce site-specific mutations phosphorylate the method described in example 2,2-1).

Getting heteroduplexes the BOTTOM.

0.5 μg single-stranded M13mp9 DNA (advanced APT (II)) and 1.5 μg of double stranded M13mp9 DNA cleaved with restriction enzymes BamH1, heated in 30 µg of a solution containing 2 pmol of phosphorylated primer (1) 10 mm Tris-HCl (pH 7.5), 0.1 mm EDTA and 50 mm NaCl at a temperature of 90oC (2 min), 50oC (5 min), 37oC (5 min) and at room temperature (10 min). To the solution was added 36 μl of a solution of 50 mm Tris-HCl (pH 8.0) containing 4 units of enzyme maple, 7 units of DNA ligase of phage T4, 0.1 mm EDTA, 12 mm MgCl2, 10 mm dithiothreitol, 0.7 mm ATP, 0,07 dATP and 0.2 mm of each of the DSTF, dTTP and dCTP, in order to stimulate the elongation of the primer. The mixture is subjected to interaction at a temperature of 20oC for 2 hours and at a temperature of 4oC for 15 hours.

The transformation is performed with the use solution, the and. After separation of colorless spots phage infect E. coli JN109 for proliferation. Then the matrix-stranded DNA is obtained from the culture supernatant of each clone. These single-stranded DNA is subjected to only the reaction T (reaction "A" and "T" in the example 3-2) dimethoxymethane using primer (2) for sequencing, followed by polyacrylamide gel electrophoresis. After drying the gel to analyze radioautography. Based on the results identify a clone with the desired mutant sequence. The culture supernatant of the clone is used to infect cells of E. coli JM109 and again inoculant on the Cup in order to move a single spot. From the obtained single spot single-stranded DNA is isolated in accordance with the above method. Using this DNA, first, determine the sequence of DNA bases by dimethoxymethane using primer (2) for sequencing, getting the clone mutated in a desirable sequence of bases. After infection of this phage clone cells JM 109 E. coli using the method of messing, described in example 2, get double-stranded DNA. This double-stranded DNA digested with restriction enzymes Xho 11 that were about 1500 base pairs, containing the gene for enhanced APT. Then elektrobudowa extracted DNA.

In addition, dimethoxymethane determine the complete sequence of the bases in relation to the thus obtained DNA, resulting in the finding that DNA is a gene enhanced APT (V). The full sequence of the bases thus obtained gene enhanced APT (V) (but containing the signal peptide from -35 to -1) is shown in Fig. 11 - 13. Derived from her amino acid sequence shown in Fig. 17 - 19.

3-2) the Design of improved APT (VI) and (VIII).

Methods similar to those described in example 3, 3-1). First, design M13mp3 (enhanced APT (II)), and then synthesize primers to induce site-specific mutations. The sequence of the bases of these primers described above, however, for constructing gene enhanced APT (VI) and gene enhanced APT (VIII) use correspondingly phosphorylated 5'-end primer (3) and phosphorylated 5'-end primer (5) (see tab. 2). Following the induction of site-specific mutations define dimethoxymethane complete sequence of the genes do advanced APT (VI) and enhanced APT (VIII).

Then these genes integrate into a vector pVY1 in accordance with the methods described in examples 4 and 5.

Example 4. Integration of the gene enhanced APT (II) in the vector pVY1.

4-1) Construction of vector pVY1.

Vector pVY1 receive as shown in Fig. 7.

A) Constructing pAdD26SV (A) N3 (N) and giving the blunt ends of the cleavage site Eco R1.

First, DNA pAdD26SV(A) N3 (purchased from Dr. Hiroshi handa at the University of Tokyo, known for abstracts in Mo1, Ce 11. Biol, 2 (11, (1982)) you unhook restriction enzyme Bgl11 (manufactured by Boehringer Mannheim-Yamanouchi Co. Co., Ltd.) the traditional way. Then DNA make obtuse traditional way using enzyme maple (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd). After treatment with phenol and precipitation with ethanol and drying under reduced pressure precipitate dissolved in sterile distilled water. After further ligating transform the reaction mixture competent cells of E. coli HB101 (manufactured Tecra Shuso, Co. Co., Ltd.). Plasmid DNA obtained from transformants expressing resistance to tetracycline, as usual.

After cleavage of these DNA with restriction enzymes BgL 1 carry out electrophoresis in a 0.7% agevalue (pAdD26SV(A) N3 (N)) the DNA of this clone with restriction enzymes Eco R1 in the traditional way, DNA make a blunt using enzyme maple, as described above. After treatment with phenol and precipitation with ethanol and drying under reduced pressure, the precipitates are dissolved in distilled sterile water.

B) slice Selection Kpn 1-BamH1 (about 2900 base pairs) of pKSV10 and the formation of blunt ends.

After DNA cleavage pKSV10 (manufactured by Pharmacia fine chemicals) restriction enzymes Kpn1 and BamH1 traditional way DNA make a blunt using DNA polymerase T4 (lab manual, pages 114 - 121). Then carry out the electrophoresis gel of 0.7% agarose emitting fragment size of about 2900 base pairs. Then the slice is subjected to electroelution for extraction of DNA

C) Constructing pVY1.

After ligating the DNA fragment obtained in (A), and the DNA fragment obtained in (B), carry out the transformation of competent cells of E. coli HB101 (described above).

From the transformants exhibiting resistance to tetracycline, the traditional way obtain plasmid DNA. After cleavage of these plasmid DNA with restriction enzymes Pst1 (manufactured by Boehringer Mannheim-Yamanouchi Co. , Ltd) carry out electrophoresis in 1.0% agarose gel. In any and about 1400 base pairs. This clone E/coli HB101 (pVY1 deposited in scientific research Institute of fermentation Agency of industrial science and technology of Japan under the registration number P-9625 (FEPM BP 2106).

4-2) Integration of gene enhanced APT (II) in the vector pVY1.

After splitting the DNA plasmids pVY1 obtained in example 4-1), restriction enzyme BgL 11 in the traditional way, by the dephosphorylation using alkaline phosphatase (manufactured by Takara Shuso. Co. Co., Ltd.). Then carry out the treatment with phenol three times. After precipitation with ethanol and drying under reduced pressure, precipitation was dissolved in sterile distilled water.

After ligation of this DNA fragment BgL 11-Xho 11 (about 1500 base pairs) obtained in examples 3, 3-1), and the competent cells of E. coli HB101 transformed with the ligation product in accordance with the method described above. Of transformants with resistance to tetracycline, get a traditional way of plasmid DNA. After cleavage of DNA with restriction enzymes (BqL 11, Pst 1) select the clone with the gene enhanced APT (II) in the vector pVY1 integrated in the desired direction, and the selection is performed based on the analysis of paintings electrovolt electrophoresis with 0.8% agarose gel, getting a clone that has a scroll fragment size of about 1500 base pairs, when the fragment BqL 11-Xho 11 are ligated with a fragment of BqL 11 plasmids pVY1, legirovannoi part 11 and Xho BqL 11 can be cut with restriction enzymes BqL 11.

Part of the plasmid DNA of these clones was additionally digested with restriction enzymes Pst1 and the DNA is subjected to electrophoresis with 0.8% agarose gel to obtain a clone having one strip of about 3400 base pairs, two bands of about 2300 base pairs, a single band of approximately 1400 base pairs and a single band of about 80 base pairs. Using this clone (plasmid pVY1-APT (II) in accordance with the laboratory manual receive plasmid DNA.

Example 5. Integration of genes enhanced APT (V), (VI) and (VIII) in the vector pVY1.

After splitting the DNA plasmids pVY1 obtained in example 4-1), restriction enzyme BqL 11 traditional way by the dephosphorylation using alkaline phosphatase (manufactured by Takara Shuso, Co., Co., Ltd.) followed by treatment (3 times) phenol, by precipitation of atenolol and drying under reduced pressure. Then the precipitate was dissolved in sterile distilled water.

After ligating the data is transforming the above competent cells of E. coli HB101. Plasmid DNA obtained from transformants expressing resistance to tetracycline, in accordance with the traditional way. After cleavage of DNA with restriction enzymes BqL11 and Pstl carry out electrophoresis in agarose gel. Through analysis of the nature of separation in agarose gel selected clones, in which the gene is enhanced APT (V) is embedded in the vector pVYI in the desired direction. First, after cleavage of these DNA with restriction enzymes BqL11 perform electrophoresis with 0.8% agarose gel with obtaining clones and get the band size is about 1500 base pairs. When a fragment BqL11-Xholl associated with the fragment BqL11 vector pVYI, part Xholl and BqL11 can be split restriction enzyme BqL11 thanks to the aforementioned configuration solitaire.

After further splitting part of the plasma DNA of these clones with restriction enzymes Pstl carry out electrophoresis at a concentration of agarose gel, equal to 0.8%, with the receipt of the clone give the band size is about 3400 base pairs, strip of about 2300 base pairs, two bands of approximately 1400 base pairs, a single band of about 800 base pairs and a single band of about 80 base pairs. Using clone (plasmid >Similarly, the vector pVYI integrate genes enhanced APT (VI) and (VIII).

Example 6. Expression of enhanced APT in CHO cells.

The plasmid pVYI - enhanced APT (VI), APT (II), APT (V) or APT (VIII) transferout DHFR-deficient CHO cells (Urlaub, et al., Proc.Natl., Acad. Sci. USA, 77(7), 4216-4224, 1980) calcium phosphate method (Graham, et al., Viroloqy, 52, 456, 1973). Discovered that transformity clone obtained on the selective medium (MEM A LPHA (-), GIBCO) in the presence of methotrexate (MTX), active APT-level from 50 to 100 units/ml (value determined fibrin/agarose plating method, described below). This clone is used for subsequent studies. As the environment for production use GIT environment (manufactured Waco, PUA chemical industry Co., Co., Ltd.), enriched with 20 international units/ml (SIGMA) Aprotinin.

Example 7. The advanced cleaning the APT from the culture supernatant of CHO cells.

The culture supernatant obtained in example 6, a partially purified by affinity column with anti-APP monoclonal antibody. Hybrid producing monoclonal antibodies, are APT, originating from human melanoma cells, traditional education is evident in ascites, extracted and purified using CELLULARONE Protein A (manufactured Biochemical industry Co., Co., Ltd.) and a buffer system MAPS for the purification of monoclonal antibodies produced BioRad the laboratories. The antibody is associated with CN3r-activated separate (production company Pharmacia fine chemicals) in relation to 4 mg per 1 ml of gel in the traditional way.

The gel with the antibody (24 ml) is mixed with four liters of culture supernatant. After careful shaking over night at a temperature of 4oC gel is loaded into a column (diameter 1.5 cm x 20 cm). Then the gel sequentially washed with 125 ml each of the following solutions (1) Tris-HCl buffer pH of 7.4 (buffer A) containing 25 international units/ml Aprotinin (manufactured by SIGMA) and 0.01% (weight/volume) tween 80, (2) buffer A containing 0.5 M NaCl, (3) buffer A containing 4 M urea, and (4) of buffer A. Enhanced APT associated with gel, elute 0.2 M glycine-HCl buffer pH 2,5, containing 25 international units/ml Aprotinin and 0.01% (weight/volume) tween 80. Active fractions restore and unite. After dialysis against 10 mm Tris-HCl buffer, pH 7,4 containing 25 international units/ml Aprotinin and 0.01% (weight/volume) tween 80, during the night dialysate concentrate in 20-30 is against 10 mm Tris-HCl buffer, a pH of 7.4, containing 0.15 M NaCl, 25 international units/ml Aprotinin and 0.01% (weight/volume) tween 80, during the night, and used for subsequent evaluation in vitro and in vivo. Finally, the specific activity increased in 3700-5000 times, and the output ranges from 36 to 42% of the activity of the PLA (defined fibrin/agarose Cup method).

This active fraction is analyzed by electrophoresis with sodium dodecyl sulfate and silver staining. In the reducing conditions at the level of 54 kilodaltons is very strong band along with several other bands. Next, the gel after the electrophoresis process of 2.5% (weight/volume) Triton X-100 and placed on a fibrin/agarose Cup for the implementation of autografi fibrin at a temperature of 37oC, thanks to which dissolved the band detected at a level of about 50 kilodaltons. On the same Cup of natural PLA appears at about 60 kilodaltons. The results indicate that the PLA, absorbed on a column with affinity for the antibody and suirvey the specified method corresponds advanced APT having a molecular weight, which is about 10,000 less than the molecular mass of naturally occurring type.

Example 8. Measurement of the specific activity uaut by measuring total protein in accordance with the method of BradFord (Bradford, Anal.Bochem., 72, 248 (1976)), using bovine serum albumin as the reference protein. The amount of antigen APT measure enzyme linked immunosorbent assay (ELISA).

Fibrinolytic activity determine the fibrin/agarose plating method and a method of dissolving film1251-labeled fibrin. Fibrin/agarose Cup is obtained by adding agar to 95% of the coagulated fibrinogen. The dissolving film1251-labeled fibrin is carried out in accordance with the description Hoyraeerts et al. (J. Biol. Chem. 257, 2912, 1982), using as a reference APT from human melanoma cells, manufactured by Biscott Inc. and standardized in accordance with the International Standard APT (Gaffuey and Curtis, Thromb. Haemostas, 53, 34, 1985).

The value of the specific activity calculated from the activity values determined by the method of dissolving film1251-fibrin, and the number of antigen specific enzyme-linked immunosorbent assay (ELISA), ranged from 300,000 to 420000 units/mg of antigen.

Example 9. Affinity advanced APT to fibrin and the activation of the fibrin

In accordance with the work Verheijen, et al./EMBOJ, 5, 3525, 1986) examine the affinity of the enhanced APT to fibrin. To fibrinogen at different concentrations of probablywon followed by reaction at room temperature for 3 minutes. Formed fibrin clot precipitious by centrifugation speed 16000 rpm for 8 minutes and determine the number of the PLA, which is not associated with fibrin, by measuring the activity of fibrin/agarose plating method. The result found that enhanced APT (VI) shows the same affinity for fibrin, the natural form. In order to investigate the degree of activation of plasminogen improved APT in the presence or absence of fibrin, carry out the following experiment. Using a tablet for titration of naturally occurring or enhanced APT is added to 0.1 M Tris-HCl buffer, pH 7.5, containing 0.3 mm synthetic substrate p-nitroanilide-Tripeptide S-2251 (H-D-Val-leulys-pNA. HCl, production CABI Inc.), 0.13 plasminogen without plasmin, 120 µg/ml DESAFIBTM(production of American Diagnostician ICA Inc.) and 0.1% tween 80, to receive the full volume of 200 μl. System support at a temperature of 37oC. after a certain period of time, measure the absorbance (optical density) at a wavelength of 405 nm using Titertech Multiscan 310 Model.

Curve "dose-effect" for amylolyticus activity enhanced APT (VI) and found in naturally occurring APT match the value in the 158 times while for advanced APT reaches 100 times. This is due to the fact that the activity improved APT (VI) in the absence of the drug DESAFIBTMbelow, approximately 1/20, than the natural activity of the PLA.

Example 10. Analysis of the enhanced APT on fibrinolytic activity in the blood of the rabbit.

Pharmakinetic by comparing the activity of naturally occurring APT (n-APT) and advanced APT the present invention on the rabbit. As is evident from Fig. 23, the improved PLA shows a significant, prolonged biological half-time of existence in the active state (natural APT shows the half-period of existence within 1-2 minutes, then as the enhanced APT biologically active within 8-15 minutes). Moreover, it is clear that the activity value is equal to 5% (the value after 30 s after injection is 100%), still remains an APT even after 60 minutes after its injection (natural APT after 60 minutes of active 0.1% of the initial).

This experiment is as follows

For tests taken Japanese white rabbit weight 2,4 kg Under anesthesia was pentobarbital the ICA and 5400 units (0.8 ml) n-APT on rabbit (values are defined fibrin-Cup method). Then collect 2.5 ml of blood from the femoral artery using a catheter at different time intervals (from 0.5 to 60 minutes) and add 1/9 volume of citric acid sodium (3.8 percent). Within 30 minutes after blood collection exercise centrifugation at low speed, separating the plasma. Using the separated plasma was measured activity of the PLA in the blood.

(1) Measurement of activity of the PLA.

After diluting 0.2 ml of plasma 3mm glacial acetic acid 16 times carry out the centrifugation of the diluted product at low speed to obtain precipitates. The precipitates dissolve 20 mm Tris-HCl, pH of 7.4, 140mm NaCl in a volume equivalent to the volume of plasma with obtaining fractions euglobulin. The activity of the PLA is determined by adding the fraction of euglobulin in a Cup of fibrin/agarose. After incubation of the plates at a temperature of 37oC for 16 hours activity APT see in the form of a plaque. A standard curve for fibrin/agarose plating method is obtained by dilution of the PLA used for the introduction of animal, up to 0.1-10000 units/ml Specific thus the activity of the PLA blood expressed in percentage, using the activity of the PLA obtained by collecting the blood in 30 seconds after LASS="ptx2">

To determine the resistance to heat enhanced APT (VI) and organic APT diluted with 50 mm Tris buffer containing 100 mm NaCl and 0.01% tween 80, pH of 7.4, to a concentration of 100 µg/ml, respectively. Each solution was incubated in boiling water (temperature 98oC) for 2-60 minutes. After cooling to determine the residual activity method fibrin-Cup. As shown in Fig. 24, reduced activity enhanced APT (VI) is insignificant compared to the reduction in the activity of natural PLA. For example, after heat treatment for 2 minutes, the activity of natural APT is reduced to 25%, whereas enhanced APT (VI) still retains activity at the level of 71%.

To study the improved acid resistance APT (VI) and natural PLA dissolved in 0.5 N. HCl solution at a concentration of 100 µg/ml, followed by sedimentation at room temperature for 30 minutes. After neutralization determine the activity of fibrin-plating method. Enhanced APT does not detect any change in activity, whereas the activity of natural PLA is reduced by 50%.

Example 12. The inhibition of the active factor stimulating lymphocytes, usovershenstvovaniia culture PPM1 1640, containing 7% amniotic calf serum and 58 μm 2-mercaptoethanol. 100 µl of the dilution loaded into a 96-well plate for tissue culture, and then add 50 ál of cell suspension containing thymocytes (2107cells/ml) from mice C3H/HeJmale in age from 4 to 6 weeks, concanavalin A (1.2 µg/ml) and 50 μl of IL-1 (4 units/ml, Aenzyme Inc), followed by cultivation for 48 hours in an incubator at a temperature of 37oC containing 5% carbon dioxide. Then add H3-thymidine at a concentration of 0.5 MK. cubic inches /20 µl/well. After culturing for 18 hours, cells are harvested on glass fiber filter and the number of3H-thymidine introduced into cells, measure the liquid scintillation counter, determining the activity of factor stimulating lymphocytes.

As shown in Fig.25, natural PLA did not inhibit the activity of factor stimulating lymphocytes, and enhanced APT significantly suppresses it. When tested only with the solvent is not observed any effect.

Example 13. Anti-inflammatory activity based on action against denaturirovannogo squirrel.

1) Getting denaturirovannyj temperature 37oC for 2-3 hours protein solution to neutralize the same amount of NaOH or HCl.

2) the Affinity of the enhanced APT (VI) to denaturirovannogo squirrel.

Method: in accordance with the methodology below, denatured protein "concatenate" with nitrocellulose film. Then measure the amount advanced APT associated with the processing of protein and nitrocellulose film, evaluating thus the affinity of the enhanced APT to denaturirovannogo squirrel.

A piece of nitrocellulose film is immersed in 20 mm Tris-HCl buffer solution, pH 7.5, containing 140 mm NaCl.

The drying. Denatured protein (50 µg/10µl) produced by drop on a piece of nitrocellulose film.

The drying. Blocking the 3% solution of gelatin.

Rinsing. A piece of nitrocellulose film is dipped in a solution of enhanced APT /mkg/ml/.

Rinsing. Add the plasminogen and the synthetic substrate S-2251, followed by incubation at a temperature of 37oC (quantitative analysis on the absorbed enhanced APT).

Measuring the absorption at 405 nm.

Results: as shown in table 3, enhanced APT on the WMD NaOH. On the other hand, enhanced APT does not show affinity to intact immunoglobulin G and albumin.

3) Activation of the enhanced APT (VI) denatured protein.

Method: plasminogen (0,0078 unit 10 μl), 100 μl of 3 mm synthetic substrate S-2251 and a variable number of TBS buffer was added to the reaction solution activator advanced APT (denatured protein Enrichment - treated fibrinogen, etc.,) at various concentrations, getting 0,275 ml of reaction solution. Enhanced APT (2,5 n/g in 25 μl) was added to the reaction solution to initiate the reaction. After interaction within a certain period of time in the reaction solution was added 2% sodium dodecyl sulphate (equimolar amount) in order to stop the reaction. By measuring the optical density (OD 405) determine the activity of the advanced APT.

Results: as shown in Fig.26, NaOH - treated albumin and HCl - treated immunoglobulin G show a pronounced activating effect of the enhanced APT. In particular, in the HCl-treated immunoglobulin G activation is strong, and the activity of HCl-treated immunoglobulin G example the stroke albumin and immunoglobulin G do not show activation.

4) Degradation of denatured protein under the action of an improved APT (VI).

Method: after interaction of denatured protein with improved APT under the same conditions as in the method described in the previous paragraph, except that in the reaction system does not add synthetic substrate S - 2251, and the amount of denatured protein is 133 µg/ml, carry out electrophoresis in polyacrylamide gel with sodium dodecyl sulfate in the presence of-mercaptoethanol.

Results: as shown in Fig.27, denatured by treatment with NaOH or HCL treatment protein leads to the disappearance of the painting EF-protein bands and the formation of degradation products, which indicates decomposition. On the other hand, the use of intact albumin did not reveal any changes in EF-picture after interaction with advanced APT, and therefore not detected any degradation of denatured protein.

1. Recombinant tissue plasminogen activator having the amino acid sequence shown in C.

where Y - Glu-Ile-Lys;

H2N - aminocore;

-COOH - carboxylic;

R is a direct bond or anal is Yunosti,

and with the following properties: fibrinolytic activity determined by the method of dissolving film125I-fibrin, the ability to be activated by fibrin and activity improved tpA in the absence of fibrin, which is lower than the activity of natural tpA, increased compared to the natural form, half-life, increased in comparison with the natural tpA, resistance to acid and heat, the ability to inhibit factor activation of lymphocytes, the ability to be activated denatured protein.

2. A method of obtaining a recombinant tissue plasminogen activator, comprising culturing host cells transformed by a recombinant DNA containing a sequence encoding the analogue of the tpA, and the subsequent purification of the target product, wherein the cultured host cell transformed with the recombinant vector containing a DNA sequence encoding a tpA under item 1.

 

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FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

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FIELD: molecular biology, medicinal industry.

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EFFECT: improved preparing method.

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FIELD: medicine.

SUBSTANCE: invention relates to new staphylokinase derivatives which represent expression products in heterogeneous system of mutant genes obtained by local gene PCR-mutagenesis ← wild type enzyme and differ from respective native staphylokinase form in one or more amino acid substitutions between 104 and 113 amino acid residues that leads to formation of RGD or KGD sequence in said site. Obtained recombinant staphylokinase derivatives (RGD/KGD-Sak), as well as variants thereof having deletions of 1-16 amino acids from NH2-terminal combine properties of thrombolitic and antocoagulating agents and are characterized by decreased polymerization ability and immunogenicity in contrast to wild type enzyme.

EFFECT: new effective agents for thrombosis preventing and treatment.

25 cl, 3 dwg, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.

EFFECT: invention makes it possible to obtain staphylokinase protein which possesses high fibrinogenic activity, with output of recombinant protein to 20% from total amount.

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SUBSTANCE: synthetic oligonucleotide primers and probes are disclosed to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus. Also the method is described to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus using such synthetic oligonucleotide primers and probes.

EFFECT: method makes it possible to perform determination of a serotype of the bluetongue disease virus with the help of PCR with a stage of reverse transcription in real time mode, and also to increase specificity and sensitivity, to reduce time to perform work aimed at determination of a serotype of the bluetongue disease virus in samples of an investigated biological material.

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EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

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FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular, to novel mutants of streptokinase. Claimed are both mutant streptokinase polypeptides and fusion proteins, possessing streptokinase activity. Claimed streptokinases are included into formulations of pharmaceutical compositions, suitable for treatment of blood circulation diseases, in particular thromboses.

EFFECT: invention makes it possible to obtain more effective mutant polypeptides with streptokinase activity in treatment of blood circulation diseases.

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FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.

SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.

EFFECT: valuable medicinal properties of activator and pharmaceutical composition.

6 cl, 6 dwg, 8 ex

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