RU2131462C1 - Dna fragment, recombinant polypeptide exhibiting antithrombin activity, method of its preparing (variants), pharmaceutical composition, recombinant vector (variants) - Google Patents

Abstract

FIELD: molecular biology, genetic engineering, pharmacy. SUBSTANCE: invention relates to preparing antithrombin polypeptide used for treatment of patients with vein thrombosis. Polypeptides are prepared by genetic engineering methods. Invention involves preparing DNA sequence encoding the end polypeptide precursor bound with leader peptide. DNA sequence is introduced into suitable expression vector containing the gene determining the resistance to antibiotic and bacterial promoter and this vector is used for transformation of cells E. coli type B. Transformed strain is cultured under conditions that are necessary for expression and the end product is isolated. Alternatively, DNA sequence is introduced into transfer vector and insect cell cotransfection is carried out with this vector and DNA baculovirus wild type. Recombinant viral DNA containing DNA sequence that encodes the end polypeptide precursor under polyendrin promoter control is isolated from the transformed cells and this sequence is used for infection of insect cell culture. DNA fragment encoding polypeptide that exhibits an antithrombin activity has the known nucleotide sequence. Recombinant expression and transfer vectors involve this nucleotide sequence. Pharmaceutical composition for treatment of patients with cardiovascular diseases comprises recombinant polypeptide showing an antithrombin activity taken at effective concentration as an active component and a pharmaceutically acceptable carrier or vehicle. EFFECT: enhanced effectiveness of vein thrombosis treatment. 10 cl, 15 dwg, 9 ex

Description

 The invention relates to polypeptides and their preparation. These polypeptides were isolated from the leech of Hirudinaria manillensis. These polypeptides have antithrombin properties.

The most well-known anticoagulant peptides obviously belong to the hirudin family. Hirudin, which was isolated from the medical leech Hirudo medicinalis, is well known in the art as a polypeptide thrombin inhibitor ^1,2 . In particular, hirudin binds thrombin through ionic interactions, thereby preventing the conversion of fibriogen to fibrin and the subsequent formation of fibrin clots. In animal studies, hirudin has been shown to be effective in preventing venous thrombosis, occlusion of anastomatic vessels, and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, low antigenicity, or its complete absence, and a short time of elimination from circulation ³ .

 Polypeptides with anticoagulating properties were isolated from various species of leeches Hirudinaria manillensis (EP-A-0347376 and WO 90/05143). This type of leech is evolutionarily more advanced than Hirudo medicinalis and therefore can synthesize anticoagulating peptides whose amino acid sequences may differ from the amino acid sequences of hirudin and its other known variants.

 The authors of this application have conducted studies to obtain drugs from leeches Hirudinaria manillensis. As a result of these studies, three new polypeptides with antithrombin activity were discovered. In accordance with this, the present invention relates to a polypeptide containing the amino acid sequence (see the end of the description), and its pharmaceutically acceptable salts.

Amino acid residues are presented in accordance with their standard three-letter code (Eur. J. Biochem. 138. 9-37, 1984). The Yaa residue is Asp or Tyr residues, and Zaa is —OH, —NH _2, or Gly-OH. Said salts may be acid addition salts. These salts can be formed by inorganic acids, such as hydrohalic acids, for example hydrochloric acid, sulfuric acid, phosphoric acid, or pyrophosphoric acid, or organic acids, such as benzenesulfonic, p-toluenesulfonic, methanesulfonic, acetic, lactic, palmitic, stearic, malic, , tartaric, ascorbic, or citric acid. These polypeptides also contain free hydroxyl groups and therefore can form sodium, potassium, calcium, magnesium or ammonium salts, or these salts can be formed with physiologically acceptable organic nitrogen-containing bases. The polypeptides may also be in the form of internal salts.

 The polypeptides of the present invention consist mainly of the above amino acid sequences (i) to (iii). Natural polypeptides isolated from Hirudinaria manillensis have the amino acid sequence (i) or (ii), where Yaa is Asp, and Zaa is —OH or partial amino acid sequence (iii). The polypeptides of the present invention can be isolated and purified for use as thrombin antagonists.

 Polypeptides can be obtained using the preceding all or part of the leader sequence. This leader sequence may be a native or foreign leader sequence with respect to the cell from which the polypeptide originates. The leader sequence has the ability to direct the secretion of the polypeptide from the cell. Both natural polypeptides of the present invention are expressed by a leader sequence, which is then cleaved. Therefore, the whole or part of the leader sequence may be present, which, in particular, is the following sequence: Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys Ile Cys Val Ser Gln Ala.

 The natural polypeptide of the present invention or its salts can be obtained by isolating the specified polypeptide or its pharmaceutically acceptable salt from tissue or leech secrets of the species Hirudinaria manillensis. In particular, the polypeptide of the present invention can be obtained by production in accordance with WO 90/05143 and purification using high pressure liquid chromatography.

The polypeptide of the present invention or its salt can also be obtained by
a) the use of a host transformed with an expression vector containing a DNA sequence encoding the specified polypeptide under conditions ensuring the expression of the polypeptide in the host;
b) isolating the polypeptide thus obtained or a pharmaceutically acceptable salt thereof.

This method basically consists in obtaining the nucleotide sequence encoding the desired polypeptide, and the expression of the specified polypeptide in recombinant organisms. The cultivation of genetically modified organisms leads to the production of the desired product with full biological activity. In addition, the present invention relates to
- to an expression vector containing a DNA sequence encoding a polypeptide of the present invention;
- to a host transformed using a compatible expression vector in accordance with the present invention;
- to a DNA sequence encoding a polypeptide of the present invention.

A host for expressing a polypeptide of the present invention can be obtained by transforming a host cell using a compatible expression vector of the present invention. This expression vector can be obtained by:
a) chemical synthesis of a DNA sequence encoding a polypeptide of the present invention;
b) insertion of the specified DNA into the expression vector.

Alternatively, the expression vector can be obtained by
a) producing and isolating a cDNA encoding a polypeptide of the present invention from mRNA of a leech of the species Hirudinaria manillensis; and
b) insertion of the selected cDNA into the expression vector.

 Thus, according to the present invention, the polypeptide is produced by obtaining a transformed host under conditions ensuring the expression of the polypeptide in this host. If the host is a eukaryotic cell, then the resulting polypeptide can be glycosylated. The resulting polypeptide can be isolated as such, or in the form of its pharmaceutically acceptable salt. Thus, the polypeptide of the present invention or its salt can be obtained in pure form.

 The polypeptides of the present invention can be modified by amino acid extension inserts at one or both ends. A polypeptide consisting of such an extended sequence should, of course, still have antithrombin activity. For example, a short sequence of up to 30 amino acid residues can be introduced at either or both ends.

 The polypeptides of the present invention can be subjected to one or more post-translational modifications, such as sulfation, COOH amidation, acylation or chemical alteration of the polypeptide chain. For example, a polypeptide having a glycine residue at its carboxyl end can be enzymatically amidated using peptidyl-glycine-α-amidating monooxygenase (PAM enzyme).

 To produce a polypeptide with antithrombin activity by using the recombinant DNA technique, a gene encoding the polypeptide of the present invention is obtained. The DNA coding sequence usually does not contain introns. This DNA is isolated and purified. The gene is inserted into an expression vector capable of producing a recombinant product. DNA sequences may be cDNA sequences. They can also be synthetic DNA sequences. A synthetic gene is usually obtained by chemical synthesis of oligonucleotides, which generally correspond to the desired gene. Then, to obtain the gene, these oligonucleotides are assembled.

 The specified gene can be constructed from six chemically synthesized oligonucleotides, each of which represents about 1/3 of the double-stranded gene DNA strand. To obtain the desired gene, the oligonucleotides are crosslinked and annealed. If desired, the gene sequence can be modified using site-directed mutagenesis by replacing one or more codons. Typically, a gene is constructed using restriction sites at each end to facilitate subsequent manipulation.

 As indicated above, a DNA sequence that encodes a leader peptide can be obtained. The specified leader peptide has the ability to direct the secretion of the polypeptide from cells in which this peptide has been expressed. The leader peptide coding sequence is typically ligated to the 5 ′ end of the DNA sequence encoding the polypeptide.

В соответствии с настоящим изобретением может быть получена ДНК-последовательность, кодирующая слитый белок, который расщепляется с высвобождением нужного полипептида. При этом может быть использована ДНК-последовательность, которая кодирует несущую полипептид последовательность, сшитую посредством отщепляемой связи с N-концом полипептида настоящего изобретения. Указанная смесь может расщепляться цианистым бромидом.Such a leader peptide may be an OmpA leader peptide, if expression is required in a bacterial host such as E. coli. When expressed in insect cells, the leader peptide may be the leader peptide of the vesicular stomatitis G virus protein (VSV-G protein). The corresponding DNA sequences encoding the leader sequences of the OmpA and VSV-G protein are shown below:

In accordance with the present invention, a DNA sequence encoding a fusion protein that cleaves to release the desired polypeptide can be obtained. In this case, a DNA sequence can be used that encodes a polypeptide-carrying sequence crosslinked by cleavage to the N-terminus of the polypeptide of the present invention. The mixture may be cleaved by cyanide bromide.

 For expression of the polypeptide, an expression vector is constructed that contains a DNA sequence encoding a polypeptide and which is capable of expressing the polypeptide in an appropriate host. Transcriptional and translational regulatory elements can be used, including a promoter for the DNA sequence, a transcription termination site, and translation initiation and termination codons. The DNA sequence is obtained in the correct reading frame so that the expression of the polypeptide occurs in a host compatible with the vector.

The expression vector typically includes a region of origin of replication and, if necessary, a selectable marker gene, such as an antibiotic resistance gene. The promoter is crosslinked with a DNA sequence encoding a polypeptide. The specified expression vector may be a plasmid. In this case, a promoter, preferably selected from P _trp and P _{lcc / lac} promoters, is crosslinked with the DNA sequence. Alternatively, the expression vector may be a virus. Such a virus may be a recombinant baculovirus in which the polyhedrin promoter is operatively linked to a DNA sequence encoding a polypeptide.

An expression vector capable of expressing a polypeptide can be obtained using standard techniques. A DNA fragment encoding a polypeptide can be inserted into the corresponding restriction site of an expression vector, for example, a plasmid vector. Recombinant baculovirus can be obtained by
(i) cloning a gene encoding a polypeptide in a baculovirus carrier vector at a restriction site located behind (in the direction of 5 ′ __ → 3 ′) a polyhedrin promoter; and
(ii) co-transfection of insect cells susceptible to baculovirus infection with a recombinant vector as defined in step (i) and wild-type intact baculovirus DNA.

 As a result, homologous recombination occurs, allowing one to obtain a recombinant baculovirus containing the polypeptide gene behind the (5 ′ __ → 3 ′) polyhedrin promoter. The baculovirus transfer vector may have a unique cloning site located behind the (5 ′ __ → 3 ′) initiating ATG polyhedrin codon. The product expressed by the resulting recombinant baculovirus is a fusion protein in which the N-terminal portion of the polyhedrin protein is fused to the N-terminus of the polypeptide. As indicated above, a cleavable bond may be at the ligation site.

 In step (ii), Spodoptera frugiperda cells are typically used as insect cells. Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as the wild-type baculovirus.

 An expression vector encoding a polypeptide is obtained in an appropriate host. Cells are transformed using a polypeptide gene. Transformed host cells are obtained under conditions ensuring the expression of the polypeptide in them. Transformed cells, for example, are cultured in such a way as to create the opportunity for the implementation of this expression. In this case, any compatible host and vector system can be used.

 The transformed host may be prokaryotes or eukaryotes. A bacterial or yeast host, for example, E. coli or S. cerevisiae, may be used. In particular, a gram-positive bacterium can be used. A preferred bacterial host is an E. coli strain of type B. Alternatively, insect cells can be used if the baculovirus expression system is suitable in this case. Such insect cells are Spodoptera frugiperda cells. In another alternative embodiment, cells belonging to a mammalian cell line can be transformed. In this case, non-human transgenic animals can be used in which the desired polypeptide is produced.

 The expressed polypeptide can be isolated and purified. In this case, a polypeptide having one of the aforementioned amino acid sequences (i), (ii) or (iii) can be obtained, which is preceded by the Met residue encoded by the translation initiation codon. Alternatively, as indicated above, a fusion protein comprising the amino acid sequence of a polypeptide of the present invention, i.e. a sequence (i), (ii) or (iii) ligated to a carrier sequence. If in the specified hybrid protein between the amino acid sequence (i) and (ii) or (iii) and the carrier sequence there is a corresponding bond, then the polypeptide having the above amino acid sequence (i), (ii) or (iii) may be isolated by cleavage of this bond with an appropriate agent.

The polypeptide of the present invention or its pharmaceutically acceptable salt can also be obtained by
(a) chemical synthesis of said polypeptide; and
(b) isolating the polypeptide thus obtained or a pharmaceutically acceptable salt thereof.

 Thus, the specified polypeptide can be constructed by chemical synthesis from individual amino acids and / or from peptides preformed from two or more amino acids in the order corresponding to the sequence of the desired polypeptide. In this case, solid-phase or solvent methods can be used. The resulting polypeptide can optionally be converted to its pharmaceutically acceptable salt.

 In solid-phase synthesis, the amino acid sequence of the desired polypeptide is sequentially constructed from the C-terminal amino acid bound to an insoluble resin. After producing the desired polypeptide, it can be isolated from the resin. When using synthesis in the liquid phase, the desired polypeptide can again be constructed from the C-terminal amino acid. The carboxyl group of the indicated amino acid residues blocked by the corresponding protecting group is removed at the end of the synthesis.

 Regardless of whether solid-state or liquid-phase techniques are used, each amino acid added to the reaction system usually has a protected amino group and an activated carboxy group. Functional side groups are also protected. After each step of the synthesis, amino protecting groups are removed. Side chain functional groups are usually removed at the end of the synthesis.

 The polypeptide can be converted into its pharmaceutically acceptable salt. It can be converted into an acid addition salt using an organic or inorganic acid. Such acid is acetic, succinic and hydrochloric acid. Alternatively, said peptide can be converted to a carboxylic acid salt, such as an ammonium salt or an alkali metal salt, for example a sodium or potassium salt.

 The polypeptide or a pharmaceutically acceptable salt thereof may be used in a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or excipient. Such a composition may be, for example, a preparation for intravenous administration (in this case, the carrier is usually sterile saline or water with an acceptable degree of purity). The polypeptide of the present invention is a thrombin antagonist and can be used in the treatment of thromboembolism, for example, in coagulation of human blood. The polypeptide can also be used to treat and prevent thrombosis and thromboembolism, including the prevention of postoperative thrombosis, in the treatment of shock conditions (for example, with septic shock or polytraumatic shock), in the treatment of consumption coagulopathy, with hemodialysis, hemoseparation and cardiopulmonary bypass. In one of the embodiments of the present invention, the polypeptide or its salt can be introduced together with a plasminogen activator, for example a tissue plasma activator.

 The dose of the polypeptide mainly depends on the specific form of administration and the goals of treatment or prevention. The size of a single dose and the administration schedule can be determined by a specialist depending on the disease, since each specialist knows methods for determining the blood factors necessary for prescribing the appropriate treatment. Basically, in the case of an injection, the therapeutically effective amount of the compounds of the present invention is in the range of about 0.005 up to 0.1 mg per kg of body weight. A dose of about 0.01 to 0.05 mg / kg body weight is preferred. An effective route of administration is intravenous, intramuscular or subcutaneous injection. Accordingly, a single dose of the composition for parenteral administration depends on the route of administration and is approximately 0.4 to 7.5 mg of the compound of the present invention. In addition to the active ingredient, said pharmaceutical composition also contains a buffer (e.g., phosphate buffer) which is used to maintain a pH in the range of about 3.5 to 7, and other components such as sodium chloride, mannitol or sorbitol to control the isotonicity of the solution. These compositions may be in lyophilized form or in the form of solutions, preferably containing antibacterial active preservatives, for example from 0.2 to 0.3% methyl or ethyl 4-hydroxybenzoic acid ester.

 The composition for external use can be obtained in the form of an aqueous solution, lotion or gel, an oil solution or suspension, or a fat-containing, or, in particular, emulsifiable ointment. A composition in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients of the present invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution (e.g. pH 4-6.5), to which, if desired, in addition to the active ingredient, anti-inflammatory agent and / or polymeric binder, for example polyvinylpyrrolidone, and / or preservative. The concentration of the active ingredient is from about 0.1 to 1.5 kg, and preferably from 0.25 to 1.0 mg per 10 ml of solution or per 10 g of gel.

 An oily form for external use can be obtained, for example, by suspending the active ingredient of the present invention or a therapeutically acceptable salt thereof in oil, optionally with the addition of a swelling agent, such as, for example, aluminum stearate and / or surfactants ( tensides) having an HLB (“hydrophilic-lipophilic balance”) value below 10, for example, fatty acid monoesters of polyhydric alcohols, for example glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan onooleat. Fat-containing ointments can be obtained, for example, by suspending the active ingredient of the present invention or a salt thereof in a suitable oily base, and optionally adding a surfactant having an HLB value below 10. An emulsifiable ointment is prepared by stirring an aqueous solution of the active ingredient of the present invention or salts thereof in a soft, greasy, spreadable base and with the addition of a detergent having an HLB value below 10. All forms intended for external use may also contain l preservatives. The concentration of the active ingredient is from about 0.1 to 1.5 mg, and preferably from 0.25 to 1.0 mg per about 10 g of the base.

 In addition to the compositions described above and their pharmaceutical analogues intended for administration directly into the human or animal body, the present invention also relates to pharmaceutical compositions and preparations for medical use outside the human or animal body. Such compositions and preparations are typically used as an anticoagulant, added to blood, circulated or processed externally (e.g., by cardiopulmonary bypass or dialysis of an artificial kidney), preservation or modification (e.g., hemoseparation). These preparations, such as stock solutions or alternative preparations in single doses, are similar in composition to the injectable preparations described above; however, the amount or concentration of the active ingredient depends mainly on the volume of blood being treated, and more precisely on the content of thrombin in it. In this regard, it should be noted that the active ingredient of the present invention in free form completely deactivates approximately 5 times the amount of thrombin by weight and is physiologically harmless even in relatively large quantities and is rapidly eliminated from the blood circulation even at high concentrations, therefore, the risk of overdose is practically absent, for example, even with blood transfusion. Depending on the specific objectives, a suitable dose of the active ingredient is from about 0.01 to 1.0 mg per liter of blood, although even exceeding the upper limit is practically safe.

 The following are examples illustrating the present invention together with the accompanying figures.

 In FIG. 1 is a chromatogram showing the results of an HPLC analysis of Example 1, P1 to P3 indicate three different peaks obtained as described in Example 1. FT corresponds to flow, and 4-AB corresponds to 4-aminobenzamidine.

 In FIG. 2 shows the elution profiles obtained in Example 2 (b) for trypsin-digested PE-P1 (A) and PE-P2 (B).

In FIG. 3 shows the nucleotide sequence of six oligonucleotides encoding most of the protein corresponding to peak 2 (P2), in which the amino acid residue at position 61 is Asp and the last amino acid of the polypeptide chain is Asn ₆₄ . Bold indicates the BalI site, which was used for further designs. At the bottom of the figure shows a method of assembly of six oligonucleotides. For subsequent manipulations, Hind III and PstI sites were used.

 In FIG. 4 shows a design scheme for the intermediate plasmid M13-P2, which is the source of the BalI-BamHI DNA fragment for all subsequent P2 constructs.

 In FIG. 5 schematically shows the construction of a new M13 recombinant, designated OMP-P2, which carries the full P2 gene linked to the OmpA leader peptide. The leader peptide sequence is shown in bold, and the blunt end of BalI and the sticky end of Hind III are underlined.

 In FIG. 6 schematically shows the construction of pFC-P2, which is a plasmid used to produce P2 protein in E. coli.

In FIG. 7 shows a general outline of the structure of the pOMPA-P2 plasmid used to produce P2 in E. coli. To obtain this new plasmid, where the P2 gene is under the transcriptional control of the hybrid promoter P _{lpp / lac} , the traditional technique of gene recombination was used. Even so, the OmpA leader peptide directs P2 secretion into the periplasm of E. coli.

 In FIG. 8 shows the nucleotide sequence and assembly of synthetic oligonucleotides used to secrete P2 from insect cells. The sequence shown in bold refers to the leader peptide of the VSV-G protein.

 In FIG. 9 schematically shows the construction of a new M13 recombinant designated VSV-P2, where the full P2 gene is linked to the leader peptide of the VSV-G protein.

 In FIG. 10 schematically shows the construction of pAc-P2, which was used as a transfer vector to the baculovirus genome. pAcYM1 is the original plasmid widely used as an acceptor of a heterologous sequence transferred to the virus.

 In FIG. 11 shows the nucleotide sequence and assembly of synthetic oligonucleotides encoding the start of a P2 chain. The ATG codon encoding the additional methionine residue is shown in bold.

 In FIG. 12 schematically shows the construction of pAcFT1, which is used for intracellular expression.

 In FIG. 13 schematically shows a new transport plasmid designated pAcFT1-P2, which contains the entire P2 sequence associated with the first 18 amino acids of the polyhedrin. This plasmid was used to transfer the heterologous sequence to the baculovirus genome.

In FIG. 14 is a schematic representation of the RACE protocol for amplification of the 3 ′ ends. In this figure, “ ^*** TTTT ...” denotes the adapter primer dT17. In each stage of the diagram, for simplicity, it is shown only how the new product obtained in the previous stage is used.

In FIG. 15 is a schematic representation of the RACE protocol for amplification of 5 ′ ends. In this figure, “ ^*** TTTT ...” and “ ^*** ” denote the adapter primer dT17 and the adapter primer, respectively. For simplicity, each stage of the diagram shows only how the new product obtained in the previous stage is used.

Example 1
The antithrombin preparation was obtained from the leech of Hirudinaria manillensis in accordance with WO 90/05143, according to the following procedures (a) and (b).

a) Extraction with acetone
Dried heads of leeches with ethanol (2920 g) were finely ground and treated with a mixture (40:60) of acetone and water (7.5 L). After homogenization by stirring at room temperature, the mixture was centrifuged for 15 min at 2700 rpm, the supernatant was drained, and the resulting precipitate was resuspended in a mixture (40:60) of acetone and water, stirred for 30 minutes and the resulting mixture was centrifuged for 15 minutes at 2700 rpm The supernatant was collected and combined, and then acidified with glacial acetic acid to pH 4.5 (volume 8.5 l). The resulting mixture was centrifuged for 15 minutes at 2700 rpm, after which the supernatant was decanted, and the pH of the solution was adjusted to pH 6.0 by adding 30% ammonia. After rotary evaporation at 35 ^° C, the pH of the concentrated solution was lowered to 1.8, the precipitated impurities were removed by centrifugation, and the crude antithrombin material was precipitated from the mixture using a 9-fold excess of acetone. Then the mixture was centrifuged, the precipitate was resuspended in acetone and centrifuged again. After that, the precipitated material was lyophilized.

b) Ion exchange cleaning
The crude antithrombin material was reduced in water, dialyzed against 10 mM ammonium acetate buffer at pH 4.0 and loaded onto a column of carboxymethyl sepharose (CM Sepharose, Pharmacia, 2.6 x 30 cm) previously equilibrated with the same buffer. Then, 100 ml was washed with the initial buffer, antithrombin-active fractions were eluted with 20 mM ammonium acetate (pH 4.5), collected and combined (1.3 L). For subsequent purification steps, the combined fractions were concentrated to 0.5 L in a Minitan apparatus (Millipore); the concentrated solution was neutralized with NaOH and then loaded onto a Q-Sepharose column equilibrated with 20 mM Tris-HCl (pH 7.0). The bound material was eluted with a linear gradient of 0 - 1 M NaCl in the original buffer. Fractions with antithrombin activity were combined, concentrated and desalted on a Superdex S-200 column, eluting with 20 mM Tris-HCl (pH 7.5) at a flow rate of 4 ml / min. The active pool from gel filtration was concentrated in a Minitan apparatus and then purified by weak ion exchange chromatography (DEAE FPLC). The active material was then loaded onto a Protein Pak DEAE-5PW column (Waters) and eluted with a 0-1 M NaCl gradient in 20 mM Tri-HCl (pH 6.5) at a flow rate of 1.0 ml / min. Active fractions were combined, analyzed for protein content and activity (specific activity: 800 ATU / mg), and lyophilized in a concentrator (Speed Vac, Savants).

 The partially purified material thus obtained (specific activity of 800 ATU / mg) was then subjected to two additional chromatography steps to obtain homogeneous polypeptides in accordance with descriptions (c) and (d) below.

c) Thrombin Sepharose
Commercial bovine thrombin (Sigma) was purified according to the procedure described by Lundblad ( ^* ), and then subjected to binding to activated Sepharose CL 6B (Pharmacia) according to the manufacturers instructions. A 1.7 ml column was equilibrated with 50 mM Tris-HCl (pH 8.3) and the lyophilized material obtained after DEAE-FPLC (reconstituted in buffer) was loaded. The column was then washed three times in the original buffer, and then in the same buffer containing 3.0 M NaCl, and again in the original buffer (each washing amounted to three times the volume of the column). The flow rate was 0.3 ml / min. The bound material was eluted with 10 ml of 0.1 M 4-aminobenzamidine in 25 mM HCl. Active fractions were collected, combined, with buffer ion exchange in 50 mm Tris-HCl pH 8.3 on a PD-10 column (Pharmacia).

 Unbound material was eluted from the column by washing in the original buffer, and material still having antithrombin activity was reloaded into the column until all this material was bound and chromatographed.

( ^* ) Lundblad, RL, 1971, Biochemistry, 10: 2501-2506
(d) RP-HPLC
The material obtained after affinity chromatography was purified by reverse phase high resolution chromatography (RP-HPLC) on a column (C4-Vidak 4.6 x 250 mm, 5 μm) using 20 mM sodium phosphate (pH 7.5) as the first eluent, and 50% acetonitrile in water, as another eluent. The antithrombin polypeptide was eluted with a linear gradient of 5% to 55% eluent B for 45 minutes at room temperature at a rate of 1.0 ml / min. The resulting chromatogram is shown in FIG. 1.

 Peaks of protein (detected at 220 nm) were collected manually, concentrated in vacuo, and chromatographed again under the same conditions.

 HPLC-purified C4 antithrombin polypeptides were analyzed for protein content, amino acid composition, N-terminal sequence, C-terminus, and their activity, which was determined using in vitro analysis (ATU / NIH test and test to determine the thrombin time ") . As a result of these analyzes, it was found that each of the three peaks of the protein had significant antithrombin activity.

 The complete amino acid sequences of the polypeptides labeled P1 and P2 in FIG. 1, was determined by N-terminal sequencing of peptides derived from tryptic and V8 protease digests. These sequences correspond to the above sequences (i) and (ii). The partial amino acid sequence of another polypeptide (P3) corresponds to the sequence indicated above (iii).

Example 2: Tryptic Digestion and Peptide Mapping of Pyridylethylated (PE) P1 and P2
(a) Reduction / Alkylation — The active fractions purified by affinity chromatography on Thrombin Sepharose (Example 1c) were combined and buffered by ion exchange in 10 mM Tris-HCl (pH 8.3) on a PD-10 column. The active pool (about 50 μg) was concentrated in a Speed-Vac centrifuge (Savant) and treated with 100 μl of 1% b-mercaptoethanol in 6 M guanidine-HCl / 50 mm Tris-HCl (pH 8.5) in the presence of nitrogen in a dark room , for 2 hours at room temperature. Then 4 μl of 4-vinylpyridine (neat) was added and the mixture was incubated for 2 hours, as described above ⁴ .

 Pyridylethylated polypeptides were first isolated from the reaction mixture by RP-HPLC on a C4 column (Vydac) (4.6 x 250 mm, 5 μm), eluted over 90 minutes with a linear gradient of 5-65% acetonitrile in 0.1% TFA, at a flow rate of 1.0 ml / min. Under such conditions, the mixture of antithrombin polypeptides is poorly separated; therefore, repeated chromatography was performed on the same column using a mixture of sodium phosphate and acetonitrile as the eluent under the conditions already described in Example 1d.

(b) Trypsin digestion and peptide mapping of PE-P1 and PE-P2
Purified PE-P1 and PE-P2 (10 and 20 μg, respectively) were digested with TPCK-treated trypsin (Sigma) in 200 μl of 1% ammonium bicarbonate (pH 8.0) in the presence of 0.2 M sodium phosphate. Trypsin was added in the ratio of the enzyme to the substrate 1:20 (w / w) and incubated for 4 hours at 37 ^° C. The digestion was stopped by freeze drying in Savant.

 Peptides obtained by tryptic digestion were separated on a C18 column (μ Bondapak, 3.9 x 300 mm, 10 μ Waters) or on a C4-Vydac column (4.6 x 250 mm, 5 μm), eluting for 60 minutes linear gradient of 5 - 65% acetonitrile in 0.1% TFA, at a flow rate of 1.0 ml / min (Fig. 2). The eluted peaks were manually collected, concentrated in Savant, and then analyzed for amino acid sequence and N-terminal sequence on a pulsed liquid phase sequencer ser. 477A (Applied Biosystems).

 The results of the indicated peptide mapping of trypsin-digested PE-P1 (A) and PE-P2 (B) are shown at the end of the description.

 Example 3: Chemical synthesis of a P2 gene.

The nucleotide coding sequence was constructed based on preferred E. coli codons ⁵ . In addition, the BalI restriction site was constructed in close proximity to the 5'-end of the synthetic gene to enable the insertion of this sequence into various expression vectors. In fact, the same synthetic gene was used to express recombinant P2 protein in bacterial or insect cells. In the case of using insect cells, methods have been developed that made it possible to obtain P2 protein in the form of a secreted or cytoplasmic product.

All manipulations with plasmid DNA were carried out in accordance with the description of Maniatis et al. ⁶
Six synthetic complementary oligonucleotides were obtained using an automated DNA synthesizer (Applied Biosystems) (the obtained sequences are shown in Fig. 3). After enzymatic phosphorylation, these 6 oligonucleotides were collected using DNA ligase and the resulting double-stranded sequence was inserted into the mp13 M13 phage vector, thereby obtaining the plasmid M13-P2, which is shown in FIG. 4. In order to be able to insert the P2 gene into the M13 vector, Hind III and PstI sites were also added to these synthetic oligonucleotides. The correctness of the nucleotide sequence was checked by the Sanger method, carried out on single-stranded phage DNA ⁷ .

 Recombinant plasmid M13-P2 was used as the source of the P2 gene for all expression vectors used in the examples.

 Example 4: Expression and secretion of P2 from E. coli cells.

To obtain secretion in the periplasm of a recombinant product, it is necessary to synthesize a P2 molecule in the form of a precursor protein. In particular, an amino acid sequence called a “leader peptide”, and responsible for efficient secretion, must be present in the NH ₂ terminal of P2 ^8.9 . This additional sequence was then cleaved in vivo during secretion by a specific E. coli leader peptidase, resulting in the correct mature sequence ¹⁰ .

The literature ^11,12 describes many examples of secretory systems. Of these, systems were selected based on a signal stimulating the secretion of the external membrane protein E. coli (OmpA) (previous publ. ¹³ ). For this, two additional complementary oligonucleotides were constructed that encode the OmpA leader peptide, which was preceded by the Shine-Dalgarno OmpA sequence, known as the sequence responsible for the efficient translation of messenger RNA ¹⁴ .

 This sequence shown in FIG. 5 also includes the beginning of a P2 gene encoding the first 10 amino acids. The presence of the BalI site allows us to bind these two synthetic fragments with the remaining P2 coding sequence, while the presence of the Hind III site located above (in the direction 3 ′ __ → 5 ′) allows us to bind them to the M13 vector. For example, a synthetic Hind III-BalI fragment was ligated to a BalI-BamHI segment from M13-P2 and inserted into M13 mp18, whereby a new plasmid designated OMP-P2 was obtained. Said new plasmid is schematically depicted in FIG. 5.

From OMP-P2, the P2 gene can be excised as a Hind III-BamHI fragment that encodes an OmpA-Shine-Dalgarno and a leader peptide, followed by a P2 coding sequence. This restriction fragment can easily be inserted into the corresponding expression vector. Theoretically, several expression systems can be used to obtain high levels of heterologous protein production in bacteria. Previously ¹⁴ , in the laboratory of the authors of this application, a system based on the P _trp promoter was successfully used. Moreover, even in the case of the selected promoter, the expression levels of this polypeptide are almost unpredictable.

Plasmid pFC33 shown in FIG. 6 has already been described in literature ¹⁴ . This plasmid carries ampicillin resistance and contains the bacterial promoter P _trp , which controls the expression of proapolipoprotein Al. After pFC33 was digested with Hind III and BamHI, a large Hind III-BamHI fragment carrying the antibiotic resistance gene and promoter was isolated and ligated to the Hind III-BamHI fragment derived from OMP-P2 encoding the P2 gene. Details of this construction are shown in FIG. 6. The isolated new plasmid designated pFC-P2 was the final plasmid for the production of P2 in E. coli.

 The subject of the present invention is the use of E. coli type B strains for expression and secretion in periplasm P2 and other antithrombin polypeptides of the present invention. Indeed, it was found that the insertion of plasmid pFC-P2 into strains of the bacterium E. coli type B allows a high level of P2 production to be obtained. In this regard, it is interesting to note that different types of E. coli strains do not work as efficiently as it might seem, so the choice of this host strain is very important for the production of bufrudin.

 For the production of P2, several E. coli type B strains can be used. Preferred strains are ATCC 12407, ATCC 11303, NCTC 10537. The following is an example of the transformation of strain NCTC 10537 using plasmid pFC-P2, and subsequent cultivation of the resulting transformant.

Competent cells of strain NCTC 10537 were obtained by the procedure using calcium chloride described by Mandel and Higa ¹⁵ . Approximately 200 μl of the preparation of these cells (at a concentration of 1 x 10 ⁹ cells per milliliter) was transformed using 2 μl of plasmid DNA (approximately 5 μg / ml). Transformants were selected on plates with L-agar containing 100 μg / ml ampicillin. Two small colonies were applied in strips with the tips of a wooden stick, with each colony being applied in the form of three strips 1 cm long on L-agar containing the same antibiotic. After a 12-hour incubation at 37 ^° C, portions of the bands were analyzed for the production of P2 protein by inoculation in 10 ml of LB medium (containing ampicillin at a concentration of 150 μg / ml) and incubated overnight at 37 ^° C. The next day, the cultures were diluted 1: 100 in M9 medium containing a similar concentration of ampicillin and incubated for 6 hours at 37 ^o C.

20 ml of this culture was centrifuged at 12,000 xg for 10 minutes at 4 ^° C. The bacterial pellet was resuspended in 2 ml of 33 mM HCl-Tris (pH 8), then an equivalent volume of a second volume of 33 mM EDTA, 40% sucrose was added, and the resulting the mixture was incubated under gentle shaking for 10 minutes at 37 ^° C. After centrifugation, cells with artificially increased membrane permeability were resuspended in 2 ml of cold water and kept on ice for 10 minutes. The resulting supernatant was isolated by centrifugation and the resulting periplasmic fraction of the bacterial cell was obtained.

Using chromogenic analysis, which is based on the inhibition of the ability of thrombin to hydrolyze the synthetic substrate S-2238 ¹⁶ , the presence of antithrombin activity was detected in the periplasmic fraction of P2-producing cells, while this activity was absent in the control periplasmic fractions.

In a similar manner, a new expression / secretion plasmid for P2 was constructed, where the P _{lpp / lac} ¹⁷ promoter was used instead of the P _trp promoter. This plasmid, designated pOMP-P2, is shown in FIG. 7. After insertion of this plasmid into E. coli type B strains, high levels of active P2 were also obtained. As the initial plasmid for the construction of pOMP-P2, the plasmid pIN-III-OmpA3 described by Ghrayb et al. ^{17 was used} . The conditions for culturing and inducing expression using isopropyl-β-D-thiogalactopyranoside (IPTG) were also similar to the conditions described previously ¹⁷ .

Example 5: Expression and secretion of P2 protein from insect cells
To secrete P2 protein from recombinant insect cells, the P2 coding sequence was coupled to a leader peptide that is well recognized by these cells. For this purpose, the leader peptide of the G protein of vesicular stomatitis virus (VSV) ^{18 was used} . Similarly (see above), a synthetic DNA sequence was obtained encoding the leader peptide of the VSV G protein, which was located behind the beginning of the P2 gene; the resulting nucleotide sequence is shown in FIG. 8. In this case, the corresponding restriction sites (Hind III, BamHI and BalI) were also constructed that allow ligation of this sequence to the remaining part of the P2 gene and the expression vector.

The synthetic Hind III-BalI fragment was ligated with a purified BalI-BamHI fragment from M13-P2 carrying the P2 gene and inserted into M13mp18 previously cut with Hind III and BamHI. As a result of this construction, a new plasmid, designated VSV-P2, which is shown schematically in FIG. 9. A BamHI-BamHI DNA fragment was excised from VSV-P2, carrying the P2 gene, ligated with the VSV leader peptide, which was then inserted into the pAcYMl ¹⁹ vector, as shown in FIG. 10. The resulting plasmid was designated pAc-P2.

 For expression in insect cells, the P2 coding sequence (VSV) must be transferred to the baculovirus genome under the transcriptional control of the polyhedrin promoter. For these purposes, insect cells were transfected together with wild-type baculovirus DNA and the pAc-P2 transfer vector. Spodoptera frugiperda cells were used as host cells. In more detail, the experiment was carried out as follows.

S. frugiperda cells were transfected with a mixture of infectious AcNPV DNA and plasmid DNA, which were separate recombinant transfer vectors, using a modification of the procedure described by Summer et al. ²⁰ One microgram of viral DNA was mixed with 25-100 μg of plasmid DNA and 0.125 M precipitated ( final concentration) calcium chloride in the presence of 20 mM HEPES buffer (pH 7.5), 1 mM disodium orthophosphate, 5 mM potassium chloride, 140 mM sodium chloride and 10 mM glucose (total volume 1 ml).

The DNA suspension was inoculated into a monolayer of 10 ⁶ S. frugiperda cells in a tissue culture dish (35 mm) and kept at room temperature for 1 hour to adsorb into the cells, and then 1 ml of medium was replaced. After incubation for 3 days at 28 ^° C., the supernatant was collected and used to produce plaques in the monolayer of S. frugiperda cells. Plaques containing a recombinant virus were identified for the absence of polyhedrosis in them using an optical microscope. Then the virus was removed from these plaques and, after purification, these plaques were used to obtain a polyhedrin-negative viral product.

The procedure described above was used to isolate a recombinant baculovirus whose genome contained the P2 gene under the control of the polyhedrin promoter and the leader peptide of the CVSV protein. This virus was used to infect S. frugiperda cells in accordance with the well-known procedure ²⁰ (with a multiplicity of infection 10). Then, infected cells were cultured in a culture with constant stirring (spin culture) or in monolayers in the presence of 10% amniotic calf serum in accordance with standard methods ²⁰ . Under both conditions, S-2238 chromogenic analysis showed the presence of antithrombin activity in supernatants of infected cell cultures.

Example 6: Expression of P2 Protein in the Cytoplasm of Insect Cells
Protein P2 can also be produced and accumulated in the cytoplasm of S. frugiperda cells. This method usually gives a higher yield of heterologous proteins, because it uses the expression signals of polyhedrin, which is an unclassified viral protein.

The method of the present invention for producing large quantities of recombinant P2 protein is based on the expression of a hybrid polypeptide, where the first 18 amino acids of the polyhedrin are connected while maintaining a reading frame with 64 amino acids of P2. The presence of the NH ₂ terminal polyhedrin sequence allows a high level of expression of ²¹ . In addition, a methionine residue is located between the polyhedrin site and the P2 sequence, which promotes the release of the P2 part during the processing of the CNBr fusion protein.

 Similarly to the previous method, a synthetic DNA fragment was first obtained, which allows the BalI-BamHI fragment from M13-P2 to be coupled to the corresponding expression vector. The new synthetic fragment shown in FIG. 11 also includes BamHI and BalI sites necessary for subsequent manipulations.

Then another carrier vector, pAcFTl, carrying the nucleotide sequence encoding the first 18 amino acids of the polyhedrin was obtained (FIG. 12). In short, the EcoRV-BamHI fragment of pAcYMl ^{19 was} replaced with a synthetic oligonucleotide containing a polyhedrin gene sequence from nucleotide -92 to nucleotide +55. The corresponding BamHI site is located after this sequence and was used to insert the complete P2 coding sequence in accordance with the scheme illustrated in FIG. 13. Using this construct, a new plasmid, designated pAcFTl-P2, was obtained, which was used to transfer the hybrid gene to the baculovirus genome.

Recombinant baculovirus was prepared as described in Example 5. S.frugiperda cells were infected according to standard procedures ²⁰ . The cultivation of infected insect cells led to cytoplasmic accumulation of the hybrid protein. This fusion protein was used as a source of recombinant P2 protein. Several methods described in the literature can be used to cleave the hybrid by CNBr ^22.23 . Application of the Olson et al. ²³ method allows one to obtain the correct P2 polypeptide sequence. This molecule showed antithrombin activity.

Example 7
To obtain the Tyr ₆₁ variant of the P2 protein, nucleotides 5 and 6 described above in Example 3 and shown in FIG. 3, was replaced by the following oligonucleotides:
Oligo 5-Tyr
5'-CGAAATCTCAGACTGAAGGTGACTTCGAAGAAATTCCGGACGAATACATCCTG AACTAGTAACTGCA 3 '
Oligo 6-Tyr
5'-GTTACTAGTTCAGGATGTATTCGTCCGGAATTTCTTCGAAGTCACCTTCA 3 '
In Oligo-Tyr-5, the TAC triplet (which is underlined) encodes a tyrosine residue and replaces the initially present GAC triplet encoding aspartic acid. Oligo-Tyr-6 was adjusted accordingly to obtain complete complementarity between the two strands. The subsequent stages leading to the expression and / or secretion of the indicated variant in insect or E. coli cells have already been described above in Examples 4-6.

Example 8
To obtain a glycine-extended derivative of a P2 protein, the oligonucleotides 5 and 6 described above (see Example 3) and shown in FIG. 3, was replaced by the following oligonucleotides:
Oligo 5-Gly
5'-CGAAATCTCAGACTGAAGGTGACTTCGAAGAAATTCCGGACGAAGACATCCTGAAC- GGTTAGTAACTGCA-3 '
Oligo 6-Gly
5'-GTTACTAACCGTTCAGGATGTCTTCGTCCGGAATTTCTTCGAAGTCACCTTCA-3 '
In oligo-5-Gly, a GGT triplet (underlined) that encodes glycine was inserted before the stop codon. Oligo 6-Gly has been adjusted accordingly to achieve full complementarity of the two strands. The subsequent steps leading to the expression and / or secretion of a Gly-extended derivative in insect or E. coli cells have already been described above in Examples 4-6.

Example 9: Cloning of cDNA P1 and P2
(a) Complete RNA from Hirudinaria manillensis heads was obtained according to the method of Cathala et al. ²⁴
(b) The reverse transcription reaction was carried out as follows:
10 μg of total RNA obtained from leech heads
1 μg dT17 adapter primer
8 μl of 5 mm mixture of dNTPs
8 μl 5X AMV buffer
H ₂ O up to 40 μl
combined and kept on ice, then mixed and the mixture was heated for 2 minutes at 65 ^o C followed by cooling with ice. After that, 10 units were added. Aranazine (Promega) and 20 units. AMV reverse transcriptase (Boehringer Mannheim) and incubated for 2 hours at 42 ^o C. Then the reaction mixture was extracted with chloroform and precipitated with isopropanol.

(c) A polymerase chain reaction (PCR) was then carried out. The following is a basic outline of this reaction:
PCR mix:
5 μl of reverse transcribed RNA
10 μl 10 x PCR buffer (Cetus / Perkin - Elmer)
16 μl dNTPs of the mixture (1.25 mM each dNTP)
2 μl MgCl ₂ 0.1 M
25-500 mm each primer
H ₂ O up to 100 μl
The reaction mixture was denatured at 95 ^° C. for 5 minutes, then 2.5 units were added. Taq polymerases (Cetus / Perkin - Elmer) and were coated on top with 80 μl of mineral oil. The reaction was carried out in a thermo cell for the DNA reaction (Cetus / Perkin - Elmer).

The following are cyclic profiles:
3 min 94 ^o C
2 min 60 ^o C
2 min 30 sec 72 ^o C 1 cycle
1 min 94 ^o C
2 min 60 ^o C
3 min 30 sec 72 ^o C 30 cycles
1 min 94 ^o C
2 min 60 ^o C
5 min 72 ^o C 1 cycle
7 min 72 ^o C
left at 25 ^o C
Residual Taq polymerase was inactivated by precipitation with phenolchloroform and ethanol and the samples were stored at -20 ^° C. Three rounds of PCR amplification were performed to obtain complete P1 and P2 cDNA sequences. The sequences of each primer are shown below. The positions at which degeneration was introduced into the oligonucleotide sequence are indicated by the alternative nucleotides shown below in the primer sequence (N means that all four nucleotides were used). Restriction sites added to facilitate the cloning of amplification products are underlined.

dT17 adapter primer:
5 'GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT 3'
XhoI SalI
Adapter primer:
5 'GAC TCG AGT CGA CAT CG 3'
XhoI SalI
Primer 3-8
5 'ATC GAA GCT TTA TAG CGA TTG TAG NGA 3'
Hind III CACC
Primer 52-56
5 'CTA AGG ATC CTT CTT CGA AGT CNCC 3'
BamHI CAA
Primer 32-37
5'ATC GGA ATT CAG TTC TGG AAA TCA GTG CGT 3 '
EcoRI
Primer 5'I
5 'CTA AGA ATT CTT CGC AAC TTA TAT GCG TT 3'
EcoRI
Primer 5 'II
5 'ATC GGA ATT CTT AAT TCA ATA TAT CTT CAT 3'
EcoRI
First round of amplification
500 pM of fully degenerated primers having a length of 3 to 8 residues and 56 to 52 residues of the P2 amino acid sequence were used as opposition primers in the PCR reaction.

Amplification of the 3'-ends of cDNA (RACE protocol), Frohman et al. ²⁵
A gene specific primer having a length of 32 to 37 residues was constructed based on the P2 nucleotide sequence determined in the first round of amplification. This primer was used together with the dT adapter primer to amplify cDNA (Fig. 14).

Amplification of the 5'-ends of cDNA (RACE protocol), Frohman et al. ²⁵
10 μg of total RNA obtained from leech heads was reverse transcribed according to the procedure described above, except that (dT17 adapter primer was replaced with 1 μg of gene specific primer (5′I) (Fig. 15). Then the reaction mixture was precipitated with isopropanol and single-stranded cDNA products were polyadenylated at its 5 ′ ends using terminal deoxynucleotidyl transferase (TdT) as follows:
22 μl cDNA
1 μl 6 mM dATP
6 μl 5X TdT buffer (BRL)
1.1 μl TdT (BRL)
These samples were incubated for 10 minutes at 37 ^o C and heated for 16 minutes at 65 ^o C. Then the reaction mixture was diluted to 500 μl of distilled water.

 10 μl of the polyadenylated product was amplified using a 10 pM dT17 adapter primer, 25 pM adapter primer, and 25 pM second gene-specific primer, located behind (in the direction 3 ′ __ → 5 ′) the first specific primer used for transcription (5 ′ II see Fig. 15).

(d) PCR Product Analysis
Amplified products were digested at restriction sites present in each primer. The digested product was gel purified and subcloned into the pUC13 vector, previously digested with the same restriction enzymes. Plasmids carrying the desired insertion were identified by restriction analysis. Plasmid DNA was sequenced using Sequenase (USB) following the recommendations of the supplier. At the end of the description, the cDNA sequences P1 and P2 thus obtained and the deduced amino acid sequences (leader sequences are underlined) are presented.

Sources of information
1) Markwardt, F. 1970, Methods in Enzymology, 19, p. 924.

 2) Markwardt, F. 1985, Biomed. Biochim. Acta. 44, p. 1007.

 3) Markwardt, F. Hauptmann, J., Nowak, G., Klessen, C., and Walsmann, P. 1982. Thromb. Haemostasis 47, p. 226.

 4) Dupont D., Keim P., Chui A., Bello R. and Wilson K., Derivatizer-Analyser User Bulletin No. 1, Applied Biosystems Inc., 1987.

 5) Grosjeans H. and Fiers W. 1982. Gene, 18, p. 199.

 6) Maniatis T., Fritsch E.F. and Sambrook J. 1982. Cold Spring Harbor, NY.

 7) Sanger, F., Nicklen, S., and Coulson, A.R. 1977, Proc. Natl. Acad Sci. USA 74, p. 5463.

 8) Blobel G. and Dobberstain B. 1975. J. Cell Biology, 67, p. 83.

 9) Pages J.M. 1983, Biochimie, 65, p. 531.

 10) Wolfe P.B. 1983. J. Biol. Chem. 258, p. 12073.

 11) Talmadge K., Stahl S. and Gilbert W. 1980. Proc. Natl. Acad Sci. USA, 77, p. 3369.

 12) Oka T., Sakamoto S., Miyoshi K., Fuwa T., Yoda K., Yamasaki M., Tamura G. and Miyake K. 1985. Proc. Natl. Acad Sci. USA, 82, p. 7212.

 13) Henning V., Royer H. D., Teather R. M., Hindennach I. and Hollenberg C. P. 1979. Proc. Natl. Acad Sci. USA, 76, p. 4360.

 14) Isacchi A., Sarmientos P., Lorenzetti R. and Soria M. 1989, Gene 81, p. 129.

 15) Mandel M. and Higa A.J. 1970. J. Mol. Biology, 53, p. 154.

 16) Krstenansky, J.K., and Mao, S.J.T. 1987. FEBS Lett. 211, p. ten.

 17) Ghrayeb J., Kimura H., Takahara M., Hsiung H., Masui Y. and Inouye M. 1984. EMBO Journal 3, p. 2437.

 18) Bailey, M.J., McLeod, D.A., Kang, C., and Bishop, D.H.L. 1989. Virology 169, p. 323.

 19) Matsuura, Y., Possee, R. D., Overton, H.A. and Bishop. D.H.L. 1987. J. Gen. Virol. 68, p. 1233.

 20) Summers, M.D., and Smith, G.E. 1987, Texas Agricultural Experiment Station Bulletin No. 1555.

 21) Luckow, V.A. and Summers, M.D. 1988, Virology, 167, p. 56.

 22) Gross E. 1967. Methods in Enzymology, 11, p. 238.

 23) Olson H., Lind P., Pohl G., Henrichson C., Mutt V., Jornvall H., Josephson S., Uhlen M. and Lake M. 1987, Peptides, 9, p. 301.

 24) Cathala, G., Savouret, J.-F., Mendez, B., West, B.L., Karin, M., Martial, J.A. and Baxter, J.D. (1983) DNA, 2.4: 329-335.

 25) Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc. Natl. Acad Sci. USA 85: 8998-9002.9

Claims (9)

1. DNA fragment encoding a polypeptide having antithrombin activity and having the following nucleotide sequence
GTTTCTTACACCGACTGCACCGAATCTGGCCAGAACTACTGCCTGTGCGTTGGTTCTAACGTTTGCGGTGAAGGTAAAAACTGCCAGCTGTCTTCTTCTGGTAACCAGTGCGTTCACGGTGAAGGTACCCCAGAGAGAGAGAGAGAGAGACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACAGAGAGAGAGAGACAGAGAGAGACGTA  2. Recombinant polypeptide with antithrombin activity encoded by the DNA fragment according to claim 1.  3. A method for expressing and secreting a polypeptide with antithrombin activity, comprising obtaining a DNA sequence encoding a precursor of a target polypeptide in which a leader peptide is linked to the N-terminus of said polypeptide, embedding the obtained DNA sequence in a suitable expression vector containing an antibiotic resistance gene and a bacterial promoter transformation of a bacterial strain with the indicated vector capable of cleaving the leader peptide used; culturing the transformed strain under ia necessary for the expression of the introduced DNA, and the selection of the target product, characterized in that the expression vector includes a DNA sequence in which a DNA fragment according to claim 1 is sequentially linked to a DNA fragment encoding a leader peptide cleaved in E. coli, and E. coli type B strain is used as a transformable bacterial strain.  4. A method for expressing and secreting a polypeptide with antithrombin activity, comprising obtaining a DNA sequence encoding a precursor of a target peptide in which a leader peptide is linked to the N-terminus of said peptide, introducing the obtained DNA sequence in the form of a recombinant construct ensuring its expression into the host cell capable of also cleaving the leader peptide used, culturing transformed cells under conditions necessary for expression of the introduced DNA, and isolating the resulting product one from the culture medium, characterized in that a DNA sequence is obtained in which a DNA fragment according to claim 1 is sequentially linked to a DNA fragment encoding a leader peptide cleaved in insect cells, the obtained DNA sequence is first inserted into the transfer vector and the cells are transfected insects with the indicated vector and wild-type baculovirus DNA, and recombinant viral DNA containing the DNA sequence encoding the precursor of the target polypeptide is isolated from the transformed cells under control of the polyendrin promoter, which is used to infect insect cell cultures.  5. A pharmaceutical composition for the treatment of cardiovascular diseases, comprising, as an active principle, an effective concentration of a recombinant polypeptide with antithrombin activity and a pharmaceutically acceptable carrier or diluent, characterized in that it contains the polypeptide according to claim 2 as an active principle.  6. Recombinant vector pFC-P2 for expression and secretion in E. coli of a polypeptide with antithrombin activity, consisting of a large Hind III - Bam HI fragment of the vector plasmid pFC33 containing the bacterial trp promoter and ampicillin resistance gene, and a DNA fragment formed sequentially interconnected Omp A - Shine - Dalgarno - a sequence, a sequence encoding an Omp A - leader peptide, and a DNA fragment according to claim 1. 7. Recombinant vector OMP-P ₂ for expression of a polypeptide with antithrombin activity in E. coli, consisting of the Hind III - Bam HI fragment of the vector plasmid pIN-III Omp A3 containing the bacterial promoter PLpp / Lac and the gene for resistance to ampicillin, and a DNA fragment formed by Omp A - Shine - Dalgarno - a sequence, a sequence encoding Omp A - leader peptide, and a DNA fragment according to claim 1, sequentially linked together.  8. Recombinant pAc-pr vector for transferring heterologous DNA to the baculovirus genome under the control of the polyhedrin gene promoter, which is the pACYMI vector plasmid, in the Bam HI site of which the heterologous DNA to be transferred is formed, formed by sequentially linked sequences encoding the leader peptide of the vesicular virus stomatitis, and a DNA fragment according to claim 1.  9. Recombinant vector pAc FTI-P2 for transferring heterologous DNA into the baculovirus genome, consisting of a synthetic DNA fragment having a polyhedrin gene sequence from nucleotide -92 to nucleotide +55, and the DNA fragment according to claim 1.

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