Recombinant staphylokinase derivative, method for production and uses thereof

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

 

The technical field to which the invention relates.

The present invention relates to new recombinant derivative staphylokinase. More specifically, the invention relates to recombinant derivative staphylokinase with significantly reduced polimerizuet ability compared with staphylokinase natural type, and bifunctionality thrombolytic and anticoagulative agent. In addition, the invention relates to the production of these recombinant derivatives staphylokinase and to their use as a thrombolytic agent.

The level of technology

Staphylokinase natural type (Sak) is a proteolytic enzyme formed under the action Lisovenko phage Staphylococcus aureus, and consists of 136 amino acid residues. Of course, by its nature Sak is not an enzyme, but it forms 1:1 complex with plasminogen (plg) in human plasma, and then this complex is activated in Sak*plm under the action of traces of plasmin (plm) on the surface of blood clots. Complex Sak*plm is a strong plasminogen activator, transforms free form plg in the active form of plm, which, in turn, catalyzes the degradation of fibrin, the main matrix of the thrombus, and thus, leads to lysis of the thrombus. Sak has fibrin specificity in the activation of plg and is more effective is but than other thrombolytic agents, dissolving old thrombi and clots, platelet-rich. Thus, the Sak is an effective and specific thrombolytic agent (D. Collen et al., Nature Medicine, 4, 279-284 (1998)). Currently, recombinant staphylokinase are manufactured by several companies of the world, but they differ from each other by the structure of genes. Thrombolytic therapy of acute myocardial infarction (mi) using recombinant staphylokinase studied D. Collen (Belgium), has completed stage II clinical trials. In the centre of Sixin (Chengdu, China) also completed stage I clinical trials for THEM, and the effect was quite satisfactory. In 1994, Shanghai medical University was designed Sak gene, performed high level expression in E. coli and completed pilot method. They were used to resolve clinical trials for the treatment of acute cerebral infarction. However, Sak, as acid protein that may have a strong antigenicity when administered to patients. Although clinical trials are not severe allergic reaction, Sak leads to high neutral titer of antibodies in most patients two weeks after injection (P.J. Declerck et al., Thromb. Haemost, 71, 129-133 (1994)). Moreover, in the study of recombinant staphylokinase discovered that staphylokinase which tend to form dimers and even polymers. The formation of polymers increases the immunogenicity of staphylokinase.

During thrombolytic therapy are often combined thrombolytic drugs with antithrombine or antithrombocyte drugs such as heparin and aspirin to ease thrombosis and prevent a second heart attack. Significant are the recent studies of thrombolytic auxiliary drugs. Functional sequences against platelet aggregation are Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD). They are competitive in contact with the receptor IIb/IIIa glycoprotein membrane associated membrane platelet aggregation, and thus preventing the binding of fibrinogen to its receptor, and prevents re-formation of a blood clot (W.H.Frishman et al., Am. Heart J., 130, 877-892 (1995); A.J.Nichols et al., Trends Pharmacol. Sci., 13, 413-417 (1992)). With the introduction of the sequence RGD/KGD in cDNA thrombolytic agent such as urokinase, when the corresponding conformational splitting, the resulting expression product will be bifunctionality thrombolytic agent and anticoagulant (J. Smith et al., J. Biol. Chem., 270, 30486-30490 (1995)). However, in clinical trials noted that the thrombolytic effect of urokinase is significantly lower than staphylokinase (50% vs. 75%). In addition, a variety of the chemical simulators based on the sequence RGD/KGD, such as tirofiban, lamifiban, depredation, orbofiban, xemilofiban, integrilin and the like, which may block the receptor IIb/IIIa. When administered in combination with thrombolytic agents, the likelihood of a second heart attack can be significantly reduced (W.H.Frishman et al., Am. Heart J., 130, 877-892 (1995); M. Verstraete et. al. 49, 856-884 (1995)).

The aim of the invention is the development of new derivatives staphylokinase, which excluded the formation of dimers and which have bifunctionality thrombolytic and anticoagulative agent, and the method of their derivation.

The invention

The present invention relates to new derivatives of staphylokinase, which excluded the formation of dimers and which have bifunctionality thrombolytic and anticoagulative agent, and the method of their production and application. In the present invention developed a new molecular structure Sak with biological structural, obtained by genetic engineering. In addition to effective and specific thrombolytic effect, the resulting products have new properties, such as low ability to polymerization and platelet aggregation. Way to obtain is simple and safe. Yield, purity and activity of the products are essentially the same as for wild-type Sak.

The brief described the e drawings

Figure 1 is an SDS-PAGE photos of various concentrations of staphylokinase natural type and recombinant staphylokinase of the present invention, in which: lane 1 is the molecular standard; lane 2, RGD-Sak (3 mg/ml); lane 3, RGD-Sak (30 mg/ml); lane 4, Sak (3 mg/ml); lane 5, Sak (30 mg/ml).

Figure 2 presents the results of tests of the sensitivity of Guinea pigs to staphylokinase natural type and RGD-Sak.

Figure 3 presents the results of testing the activity of RGD-Sak against platelet aggregation.

Detailed description of the invention

Staphylokinase natural type represents an ellipsoidal molecule, which includes α-helix containing 12 amino acid residues, covered with two β-sheets, containing respectively 5 and 2 β-round, extending from amino acid residue 20. Twenty amino acids with terminal amino groups freely protrude outwards, which hardly can be inferred from the crystal structure. For Sak, there is an apparent asymmetry in the hydrophobic region and the active region is mainly in the hydrophilic side (..Zhan et al., Acta Cryst., D52, 564-565 (1996); A. Rabijns et al., Nat. Struct. Biol., 5, (1997)). To determine the binding regions of the dimer Sak was performed molecular modeling Association with the use of the software GRAMM V1.03, bit is spent I.A. Vakser (Rockefeller University, USA) based on the crystal structure of Monomeric Sak according to x-ray diffraction. When using single molecule Sak as a receptor and the other Sak molecule as a ligand was searched connecting region in the receptor Sak relatively ligand Sak. Were found 10 structures of the complex obtained by combining, with a common high resolution combination settings that are recommended by the author. These simulations are carried out using automated graphical desktop SGI 02.

Table 1A
Combination options with a common high-resolution
Matching method (generic/spiral)method = birth
The grating pitchthis= 1,7
Repulsion (attraction always 1)ro = 30
Double spacing of attraction (the share of individual interval)share = 0
Type of potential interval (the radius of the atom, the grid step)crang = radius of the atom
Display (black and white, gray)ccti = gray
Representation (hydrophobic part)crep = all
The number with which losowanie to output max = 10
Angle rotations, degrees(10, 12, 15, 18, 20, 30, 0 - no rotations.)ai = 10

Analysis of the electrostatic potential and hydrophobicity showed that Monomeric Sak is significantly asymmetric in the hydrophobic region. Silence and researcher. the statistical method of mutagenesis showed that the amino acids that define the activity, mainly located on the hydrophilic side (K. Silence et al., J. Biol. Chem., 270, s-27198 (1995)). On the hydrophobic side of the Sak has two major hydrophobic region (TH), which are located in the area 47-56 (GO) residues and 104-113 (GO), respectively, of which HO is more hydrophobic. In the structural model constructed dimer interaction between the hydrophobic areas is very important, and there are two types of binding - GO-GO and GO-GO. because HE is closer to the active region, the area of one molecule Sak will be closed when two molecules Sak contact with each other by the type HO-HO, probably, preserving the activity of only one molecule Sak. When linking two molecules Sak with each other by the type HO-HO, apparently, the activity will not be much change.

Typically, the surface section of the interaction of protein molecules is between 600 and 1300 square Angstrom (A2), with each molecule provides 10-30 pin is chirouse residues. However, on the surface of section is the so-called "hot spot", where only 3-5 amino acids account for about 80% of the binding energy. Modification of these residues can cause a significant decrease in binding capacity of the complex (C. Li et al.. Science, 270, 1657-1660 (1995)). Thus, regardless of the specific nature of the binding of the dimer, the formation of dimers will likely be prevented, when you change the key binding residues GO. Phenylalanine (Phe 111), strongly hydrophobic amino acid, is situated in the Central area GO and is far away from the active region. In a preferred embodiment of the invention the acid is replaced by a strongly polar amino acid, asparagine (Asp), to disrupt the hydrophobic effect. It is expected that the mutant will remain active. In addition, since the peptide sequence RGD/KGD can inhibit platelet aggregation, and the hinge region inside βlists is quite free conformation, in the present invention lysine (Lys 109) is replaced by arginine (Arg), which leads to the sequence RGD-Sak, or Lys 109 do not change, which leads to a sequence KGD-Sak. Amino acid sequence of the invention RGD-Sak shown in SEQ ID No. 1, and the amino acid sequence KGD-Sak shown in SEQ ED No. 3.

In another aspect of the present invention is the method of obtaining derivatives staphylokinase of the present invention, which includes: preparing a DNA fragment containing at least part of the coding sequence staphylokinase that provides its biological activity; implementation of in vitro (outside the body) targeted mutagenesis of the DNA fragment to replace one or more of the codons of amino acids natural type codon (codon) other amino acids (other amino acids); the cloning of the mutated DNA fragment into a suitable vector; transformation or transfection of a suitable host cell with the recombinant vector; culturing the host cell under conditions suitable for expression of the DNA fragment; and extraction and purification of the desired derivatives staphylokinase from the culture medium.

Targeted mutagenesis can be carried out by polymerase chain reaction (PCR). In a preferred embodiment, the first amplification is performed using a reverse primer, and a mutant primer, with plasmid pST-Sak as a matrix. After extraction and purification by electrophoresis on agarose gel is used as a primer it amplificatory double-stranded fragment to a second amplification reverse primer, again using plasmid pST-Sak as a matrix. After purification the fragment is used as the matrix in the third signal amplification for mobile and, together with the direct primer and reverse primer. Product Satupaitea Klenow fragment, cleaved with EcoRI and BamHI, legasuite with plasmid pUC19 cleaved EcoRI/BamHI and transformed into the strain E. coli JM109. A positive clone selected by screening the analysis of cleavage, and the presence of the desired mutations in the expected position is confirmed by analysis of the nucleotide sequence. The sequence analysis is performed using the analyzer sequence ABI 377 firms Genecore Biotechology Co. Then gene RGD-Sak is removed by splitting EcoRI and BamHI and legasuite in the corresponding site of the expression vector pLY-4.

direct primer: 5'-CGCGAATTCATGTCAAGTTCATTCGAC-3'

reverse primer: 5'-CGCGGATCCTTATTTCTTTTC-3'

mutant primer (I): 5'-TAAATCTGCGACGACGTCACCACGTTCTGTTATAGG-3' (introduced website > PST, used to design the gene RGD-Sak) mutant primer (II): 5'-ATCTGGGACGACGTCACCTTTTTCTG-3' (introduced website > PST, used to design the gene KGD-Sak).

The above method can be obtained by different DNA fragments comprising the nucleotide sequence encoding the derived staphylokinase of the present invention. These fragments include the nucleotide sequence represented in SEQ ID No. 2 or SEQ ID No. 4.

Recombinant plasmid expression obtained by ligature DNA fragments of the invention in the vector expr is hurt. The present invention is not limited to any particular expression vector, provided that it can recombine with the above DNA fragments with the formation of a plasmid suitable for expression. In the preferred embodiment, uses a prokaryotic expression vector, for example, pLY-4, T7 expression system, the expression system PL, etc.

The above recombinant expression vector may be introduced into a suitable cell host using traditional techniques. The present invention is not limited to any particular cells of the host, provided that they can Express the recombinant expression vector. In a preferred embodiment of the invention uses a strain of E. coli, for example, C, JF1125, series JM, DH5α and so on

The expression product of the present invention is constructed in cells in the form of inclusion bodies. The desired product can be isolated from inclusion bodies and purified by conventional methods, for example, by the destruction of cells by French press, and collect the inclusion bodies by centrifugation.

Details of all the basic laboratory techniques of molecular biology in the above embodiments, the implementation described in Laboratory guide to molecular cloning.

In addition, nucleic acid and soo is related polypeptides of the present invention include sequences which differ from the sequences shown in SEQ ID No. 1-4, silent mutations. These modifications sequences include, for example, nucleotide substitutions that do not change amino acid sequence (for example, other codons for the same amino acids or degenerate sequence). Amino acid sequences homologous polypeptides may differ from the sequences SEQ ID No. 1 or SEQ ID No. 3 by the fact that one or more amino acid residues are inserted, deleted or replaced by other excellent amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative substitution of amino acids that have a negligible effect on the laying and/or activity of protein molecules; small deletions, typically of approximately 1 to 30 amino acids; small extension at the amino - or carboxyl end, for example, meinenemy balance aminocore; small connecting peptide having a length of about 20-25 residues; or a small extension that can facilitate the purification by changing the total charge, or who have other functions, for example, poly-his-tag tract, antigenic epitopes or binding domains.

The authors of the present invention found that the functions of the thrombolytic agent and anticoagulant products expression remain almost unchanged when you delete the coding sequence of 6-10 amino acids NH2-end of RGD-Sak or KGD-Sak by deletion mutations; the products of expression of lose thrombolytic activity, when you delete the coding sequence of 10-15 amino acids with NH2-end; this bifunctionality products expressii saved when you delete the coding sequence of the 15 iminoxyl and serine (Ser) at position 16 is replaced with lysine (Lys).

Example 1. Sample receipt and determination of properties of RGD-Sak

A. Identification of r-Sak natural type

Staphylokinase r-Sak natural type, obtained in the laboratory of the applicant (970923), has a purity of above 98% and stored at -70°C.

Reductive and non-reductive SDS-PAGE carried out according to the method of Laemmli (see Laboratory guide to molecular cloning).

Downloadable buffer solution contains 0.0625 mol/l Tris-HCl (pH of 6.7), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% mercaptoethanol and 0.001% bromophenol blue (dye).

Handling and loading of sample vials with liofilizirovannam sample (3 mg/vial, stored at -70°C for more than 3 months) dissolved in 3 ml of distilled water. Load volume is 10 ál. Staining of the gel provide bright blue Coomassie R-250 or silver dye.

Scanning Polo protein is in the gel is performed using ImageMaster® VDS (firm Pharmacia), and the amount of protein contained in each band, analyze, using the supplied software.

After electrophoresis the gel is stained bright blue Coomassie, and appear dense bands at positions corresponding to their relative molecular masses of approximately 15.5 kD, 31 kD and 46 kD and 62 kD (1 kD = 1000 y.e.).

The activity determination using the inverted method on the plate casein gel.

The above gel sequentially washed thoroughly with a 2.5% solution of Triton X-100 and distilled water, placed on a plate of agar gel (1% agar)containing fibrinogen, human plasminogen, and thrombin and incubated at 37°C for 8 hours Transparent strip lysis appear in positions corresponding to the above molecular mass, this suggests that r-Sak natural type tends to form during storage polymers anti-SDS, which is stable and active.

b. Molecular modeling of the dimer of staphylokinase and sample specimens

The simulation is carried out on an automated graphical desktop SGI 02 using the software molecular Association GRAMM V1.03 developed I.A.Vakser (Rockefeller University, USA).

To determine the binding region of the dimer Sak Association of molecules Sak model for prog is the Amma GRAMM V1.03 based on the crystal structure of Monomeric Sak according to x-ray diffraction.

The present invention replaces phenylalanine (Phe 111) asparagine, strongly polar amino acid to disrupt hydrophobic interactions. It is assumed that the mutant retains its activity. In addition, since the peptide sequence RGD can inhibit platelet aggregation, and the hinge region inside βlists is quite free conformation, Lys 109 is replaced by arginine (Arg)to get the RGD sequence.

C. Cloning of the gene RGD-Sak and construction of prokaryotic expression plasmids

Using as matrix pST-Sak, spend the first amplification with direct primer and mutant primer (I), shown below. After extraction and purification from agarose gel of the amplified fragment 351b, the latter is used when implementing a second round of amplification with reverse primers, shown below, again using as matrix pST-Sak. After cleaning, using a fragment 408b as a matrix, spend a third amplification with direct primer and reversed primer. Product Satupaitea Klenow fragment, cleaved with EcoRI and BamHI, legasuite with pUC19 and transformed. A positive clone selected by analysis of the cleavage, and the presence of the desired mutations is confirmed by analysis of the nucleotide sequence. The sequence analysis is performed using anal the jam sequence ABI 377 firms Genecore Biotechology Co. Then gene RGD-Sak is removed by splitting EcoRI and BamHI and legasuite in the corresponding site of the expression vector pLY-4.

direct primer: 5'-CGCGAATTCATGTCAAGTTCATTCGAC-3'

reverse primer: 5'-CGCGGATCCTTATTTCTTTTC-3'

mutant primer (I): 5'-TAAATCTGCGACGACGTCACCACGTTCTGTTATAGG-3' (introduced website > PST).

All enzymes, modifying nucleophilic acid, sold by firms GIBCO BRL and Promega. The oligonucleotides were synthesized in the Group of DNA synthesis at John Hopkins University (USA).

The strain JM109 E.Coli and pUC19 available in the laboratory of the applicant. Strain JF1125 E.Coli and the prokaryotic expression vector pLY-4 courtesy of Professor Jing-Juan Liu from the Institute of biochemistry, Chinese Academy of Sciences (China). Plasmid pST-Sak constructed in the laboratory of the applicant (Chinese patent No. 94 1 12105.4).

The desired gene are ligated in pLY-4 and transforming the strain JF1125 E.Coli. Plasmid receive and identify by appropriate analysis of cleavage. Get a specific passage that confirms the positive clone.

Strain JF1125 E. coli transformed with pLY-4-RGD-Sak, cultivated in culture medium MSA at 30°to reach the value of optical density (OD 600), equal to 0.6. Then increase the temperature to 42°and the cultivation continued for further 3 hours to induce expression. The expression product analyzed by SDS-PAGE. After electrophoresis half cont the KTA painted bright blue Coomassie. There is a dense band at the position corresponding to molecular mass of about 15.5 kD on the track of the product of the induced lysis of bacterial cells, which make up more than 50% of the amount of proteins of bacterial cells, according to the assessment by scanning. The other half is placed on a plate of agar gel after removal of sodium dodecyl sulfate and incubated at 37°within a few hours. Appears a transparent region corresponding to the molecular mass of 15.5 kDa. In other words, in this position, the decomposed casein, suggesting that RGD-Sak has fibrinolytic activity. After the destruction and centrifugation of cells found that the bandwidth of 15.5 kD mainly present in the sediment and barely observed in the supernatant; this indicates that the expression product exists in the form of inclusion bodies.

d. Inducible expression in engineered strains

Constructed strains are screened for high expression level (e.g., recombinant protein expression is more than 50% of the amount of cell proteins). With the selected strain are low-density fermentation in a 10-liter fermenter. After 3 hours of thermally induced cultivation, cells are untwisted, washed in phosphate-buffered saline and stored at -70°s to use. 10 l cool the tours get 80 g of wet cells. These wet cells suspended in phosphate buffer, destroy in the high-pressure homogenizer and centrifuged. Select samples for SDS-PAGE. The results show that the desired protein is present on the track of sediment, with densely stained band at the position corresponding to molecular mass of 15.5 kDa, and any staining hardly observed at the position corresponding to the liquid above the sediment; this suggests that RGD-Sak mainly exists in the form of inclusion bodies.

E. Isolation, solubilization and resaturate tel inclusion

After the destruction by compression of 80 g of cells engineered strain centrifuged at a speed of 10,000 rpm and get 20 g of inclusion bodies. After washing in phosphate buffer (FB, 0.05 mol/l) of the inclusion body was dissolved in a solution containing 0.1 mol/l FB with a pH of 5.0, 6 mol/l guanidine hydrochloride, 0.5% of β-mercaptoethanol, and incubated at room temperature until the solution becomes transparent. After ultracentrifugation at a speed of 30000 rpm./min the precipitate discarded, and the supernatant diluted to renaturation in FB (0.1 mol/l, pH 5.0) and 0.5% β-mercaptoethanol.

f. Chromatography on columns of Sephadex G-10 and S-Sepharose FF

After concentration by ultrafiltration (molecular weight 1000, Millipore), the supernatant was filtered through a column of Sephadex G-10. The filtrate under the Ute on a column of S-Sepharose FF, pre-equilibrated with ten times the amount (by volume gaskets) phosphate buffer (0.1 mol/l, pH 5.0). To control the flow rate and peak detection of the protein using a chromatograph (Waters). After loading the column was washed to baseline (no signal) phosphate buffer and elute the product with a solution of sodium chloride with a gradient of 0-1 mol/l Select erwerbende faction. The distribution of the desired protein analyzed by the method of SDS-PAGE and determine the concentration by the Bradford method (reagents purchased by the company Bio-Rad).

All operations chromatographic analysis are common for specialists in this field of technology.

g. Identification and purity determination of molecular mass

The sample analyzing method of SDS-PAGE in accordance with the Laboratory reference molecular cloning. After staining of the gel bright blue Coomassie R-250 being scanned on the device Imagemaster VDS (firm Pharmacia)to determine the purity and molecular mass of the protein. Accordingly, it has been found that the purity is more than 95%, and a molecular weight equal to approximately 15.5 kD.

h. Determination of biological activity

To determine the biological activity have been analyzing the number of plaques on casein gel (A.G. Pipemo et al., J. Exp. Med. 48(1), 223-234 (1978)) and the method of chromogenic substrate H.R. Lijnen et al., J. Biol. Chem., 266, 11826-11832 (1991)). The specific activity is about 90000-100000 HU/mg To determine units see O.Q. Tang et al., Drug Biotechnol, (Chinese), 4(1), 1-4 (1997).

i. The definition of constants Km and Kcat for complexes Sak-plasmin and RGD-Sak-plasmin

Stand Sak or RGD-Sak (2 µmol/l) with 2 µmol/l plasminogen, respectively, in 0.1 mol/l phosphate buffer (pH 5.0) at pH 7.4 and temperature 37°C for 30 min to form complexes with plasmin. Then select a catalytic amount (5 nmol/l) complex for the interactions within 0-10 min in the reaction systems listed below, in 0.1 mol/l FB at pH 7.4 and temperature 37°C, the optical density (405 nm) every 30 seconds concentrations of plasminogen are averaged according to the three tests.

The reaction systemThe final concentration
Sak-plasmin (RGD-Sak-plasmin)5 nmol/l
Chromogenic substrate1 mol/l
The plasminogen1-30 µmol/l

Activation of plasminogen complex RGD-Sak-plasmin is described by the equation of Michaelis-Menten (.1b).

Table 1b.
Mapping constants of the enzymatic kinetics of plasminogen activation by complex RGD-Sak-plasmin and Sak-plasmin
Km, umol/lKcat, with-1Kcat/Km
Sak-plasmin6.42 per1,030,16
RGD-Sak-plasmin12,501,410,11

j. Determination of ability to polymerization

As a control, use the Sak natural type. Samples dissolved in physiological solution. Tests carried out at two concentrations of protein (30 mg/ml (high) and 3 mg/ml (low). The solution was kept at room temperature. Samples taken every 24 h and analyzed by electrophoresis.

At both concentrations of the protein the ability to polymerization RGD-Sak was significantly lower than the Sak natural type (figure 1).

k. Sensitivity test on Guinea pigs

To test the sensitivity of the recombinant staphylokinase natural type and mutant RGD-Sak was dissolved in sterile saline at a concentration of 2500 units/ml For each injection take the intact capsules to prepare a sterile manner fresh solutions. Healthy Guinea pigs (20) randomly distributed into two groups, 10 animals each. This marine St is the Russia intraperitoneally administered 0.15 mg/kg of r-Sak or RGD-Sak every other day for three times. The first and second intravenous exposure dose of 0.3 mg/kg performed respectively at 14 and 21 days. Two healthy Guinea pigs, which did not enter the injection, injected with 0.3 mg/kg of the above samples and see a similar reaction to exclude pharmacological and pathological interference associated with the samples.

In the group with injection of r-Sak natural type: 8 Guinea pigs showed positive reaction of the class IV and 2 Guinea pigs showed positive reaction of the class II.

In the group with injection of RGD-Sak: 2 Guinea pigs showed positive reaction of the class I and the other animals had no detectable reaction.

The reaction of class I: soft cough

Reaction class II: coughing, tremors

The reaction of the class III: strong tremors

The response class IV: convulsion, spasm, incontinence of urine and feces, shock fatal.

The content of antibodies in the serum of Guinea pigs immunized within 1-3 weeks, determined by the method of enzyme linked immunosorbent assay (ELISA), in which the antigens used wt-Sak and RGD-Sak, respectively. In the first week the number of antibodies against any antigen was little. During the second week the number of antibodies groups wt-Sak (n=10) increases to 1:800, while the number of antibodies groups RGD-Sak (n=10) increases to 1:V third week antibody group wt-Sak is increased to 1:3200, while in the group of RGD-Sak is equal to 1:400. Thus, the immunogenicity of RGD-Sak is significantly reduced compared with the immunogenicity of wt-Sak (pigv).

The above results indicate that the immunogenicity of RGD-Sak is significantly reduced compared with the immunogenicity of wt-Sak.

l. Analysis of inhibition of platelet aggregation

Slowly centrifuged (acceleration 150 g, 10 min) fresh blood with the addition of 1/10 volume of a solution (110 mmol/l) anticoagulant - citrate to obtain a plasma enriched with platelets (SWEAT). Add RGD-Sak to SWEAT until the final concentration of 2 µmol/l, and the mixture was incubated 2 min at 37°and With continuous stirring. Then add the adenosine diphosphate (ADP) to a final concentration of 2 µmol/l as an inductor. The degree of platelet aggregation determine within 5 minutes dual unit of platelet aggregation (CHRONO-LOG560). As a control using samples of r-Sak natural type (2 µmol/l) and saline. Adenosine diphosphate buy the company Sigma, and other reagents of analytical purity produced in China.

Found that the degree of aggregation in the group of RGD-Sak (5%±2%, n=3) significantly lower than aggregation in groups r-Sak (58%±3%, n=3) and saline solution (59%±3%, n=3). This suggests that RGD-Sak has a strong potential in Engibarov the Institute of platelet aggregation called ADP (figure 3).

m. Thrombolytic analysis on animals

Thrombolytic analysis on animals carried out with RGD-Sak, obtained in the present invention, checking that RGD-Sak retains the same thrombolytic properties that Sak natural type. For the introduction of RGD-Sak was dissolved in sterile saline, as described in section k of this example.

(i) Treatment of thrombosis of the femoral artery of the experimental rabbits RGD-Sak

In each group processed RGD-Sak handled Sak natural type and in the control group without treatment, contained 6 animals. Using arteriography shows that before treatment the middle segment of the femoral artery was not seen. When you re shooting in 60 minutes after intravenous injection of 0.1 mg/kg RGD-Sak femoral artery becomes completely filled with the restoration of the cycle of blood, which is consistent with the effect in the group Sak natural type, while in the control group, the femoral artery becomes full.

(ii) Treatment of givemy experimental rabbits using RGD-Sak

In each group processed RGD-Sak handled Sak natural type and in the control group without treatment, contained 6 animals. After 4 h after intraocular injection of 10-20 µg RGD-Sak observed lysis of thrombus givemy, and red blood cells wasp is given and form a boundary surface with aqueous fluids. Intraocular blood tumor is removed after 24 hours This is consistent with the effect in the group Sak natural type. However, in the control group giveme not significantly changed.

(iii) thrombolytic therapy using RGD-Sak is safe and effective in the treatment of acute myocardial infarction in experimental dogs, caused by thrombosis of a coronary artery. In the experimental group, consisting of 6 animals, dogs received a dose of RGD-Sak (0.3 mg/kg body weight) via intravenous infusion; in the control group consisting of 6 animals, dogs received saline instead of RGD-Sak, via intravenous infusion. Before and after entering a dose, do coronary arteriography. To enter the dose found that the left anterior descending branch of the coronary artery is empty or not completely filled in animals of both groups. When conducting arteriography after 30 minutes of treatment found that the left anterior descending branch again filled, and the animals in the experimental group survive. As for the control group, not completely filled arteries there are no noticeable changes, and animals die within a few hours.

(iv) thrombolytic therapy using RGD-Sak is safe and effective in the treatment of acute cerebral infarction at the Expo is imentally pigs, caused by thrombosis of intracranial arteries. In the experimental group, consisting of 6 animals, pigs received a dose of RGD-Sak (0.2 mg/kg body weight) via intravenous infusion; in the control group consisting of 6 animals, pigs received saline instead of RGD-Sak, via intravenous infusion. Before and after entering a dose, do intracranial DSA arteriography. To enter the dose found that some of intracranial arteries are not completely filled in animals of both groups. When angiography after 30 minutes of treatment found that intracranial artery again filled, and the animals in the experimental group survive. As for the control group, not completely filled intracranial arteries there are no noticeable changes, and animals die a few days later.

Example 2. Sample receipt and determination of properties KGD-Sak

A. Identification of r-Sak natural type

Staphylokinase r-Sak natural type, obtained in the laboratory of the applicant (970923), has a purity of above 98% and stored at -70°C.

Reductive and non-reductive SDS-PAGE carried out according to the method of Laemmli (see Laboratory guide to molecular cloning).

Downloadable buffer solution contains 0.0625 mol/l Tris-HCl (pH of 6.7), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% Merck is patanol and 0.001% bromophenol blue (dye).

Handling and loading of sample vials with liofilizirovannam sample (3 mg/vial, stored at -70°C for more than 3 months) dissolved in 3 ml of distilled water. Load volume is 10 ál.

Staining of the gel provide bright blue Coomassie R-250 or silver dye.

Scanning of protein bands in the gel is performed using ImageMaster® VDS (firm Pharmacia), and the amount of protein contained in each band, analyze, using the supplied software.

After electrophoresis the gel is stained bright blue Coomassie, and appear dense bands at positions corresponding to their relative molecular masses of approximately 15.5 kD, 31 kD and 46 kD and 62 kD (1 kD = 1000 y.e.).

The activity determination using the inverted method on plates casein gel

The above gel sequentially washed thoroughly with a 2.5% solution of Triton X-100 and distilled water, placed on a plate of agar gel (1% agar)containing fibrinogen, human plasminogen, and thrombin and incubated at 37°C for 8 hours Transparent strip lysis appear in positions corresponding to the above molecular mass, this suggests that r-Sak natural type tends to form during storage polymers anti-SDS, which is stable and active.

<> b. Molecular modeling of the dimer of staphylokinase and sample specimens

The simulation is carried out on an automated graphical desktop SGI 02 using the software molecular Association GRAMM V1.03 developed I.A.Vakser (Rockefeller University, USA).

To determine the binding region of the dimer Sak Association of molecules Sak model program GRAMM V1.03, based on the crystal structure of Monomeric Sak according to x-ray diffraction.

The present invention replaces phenylalanine (Phe 111) asparagine, strongly polar amino acid to disrupt hydrophobic interactions. It is assumed that the mutant retains its activity. In addition, since the peptide sequence KGD can inhibit platelet aggregation, and the hinge region inside βlists is quite free conformation, believe that thrombolytic activity will not be affected.

C. Cloning of the gene KGD-Sak and construction of prokaryotic expression plasmids

Using as matrix pST-Sak, spend the first amplification with direct primer and mutant primer (II)shown below. After extraction and purification from agarose gel of the amplified fragment 351b, the latter is used when implementing a second round of amplification with reverse primers, shown below, again the IP is using as matrix pST-Sak. After cleaning, using a fragment 408b as a matrix, spend a third amplification with direct primer and reversed primer. Product Satupaitea Klenow fragment, cleaved with EcoRI and BamHI, ligyrus with pUC19 and transformed. A positive clone selected by analyzing the splitting, and the presence of the desired mutations is confirmed by analysis of the nucleotide sequence. The sequence analysis is performed using the analyzer sequence ABI 377 firms Genecore Biotechology Co. Then gene KGD-Sak is removed by splitting EcoRI and BamHI and legasuite in the corresponding site of the expression vector pLY-4.

direct primer: 5'-CGCGAATTCATGTCAAGTTCATTCGAC-3'

reverse primer: 5'-CGCGGATCCTTATTTCTTTTC-3'

mutant primer (II): 5'-ATCTGGGACGACGTCACCTTTTTCTC-3' (introduced website > PST).

All enzymes that modify nucleic acids, are acquired by firms GIBCO BRL and Promega. The oligonucleotides were synthesized in the Group of DNA synthesis at John Hopkins University (USA).

The strain JM109 E.Coli and pUC19 available in the laboratory of the applicant. Strain JF1125 E.Coli and the prokaryotic expression vector pLY-4 courtesy of Professor Jing-Juan Liu from the Institute of biochemistry. Chinese Academy of Sciences (China). Plasmid pST-Sak constructed in the laboratory of the applicant (see Chinese patent).

The desired gene are ligated in pLY-4 and transformed into strain IF 1125 E. coli. Plasmid and get the ID manually is ciruit by an appropriate analysis of cleavage. Get the characteristic fragment that confirms the positive clone.

Strain JF1125 E. coli transformed with pLY-4-KGD-Sak, cultivated in culture medium MSA at 30°to reach the value of optical density (OD 600), equal to 0.6. Then increase the temperature to 42°and the cultivation continued for further 3 hours to induce expression. The expression product analyzed by SDS-PAGE. After electrophoresis half of the product is painted bright blue Coomassie. There is a dense band at the position corresponding to molecular mass of about 15.5 kD on the track of the product of the induced lysis of bacterial cells, which make up more than 50% of the amount of proteins of bacterial cells, according to the assessment by scanning. The other half is placed on a plate of agar gel after removal dodecylsulfonate sodium and incubated at 37°within a few hours. Appears a transparent region corresponding to the molecular mass of 15.5 kDa. In other words, in this position, the casein is decomposed, suggesting, KGD-Sak has fibrinolytic activity. After the destruction and centrifugation of cells found that the bandwidth of 15.5 kD mainly present in the sediment and barely observed in the supernatant; this indicates that the expression product exists in the form of inclusion bodies.

d. Inducible expression in sconst wirowanych strains

Constructed strains are screened for high expression level (e.g., recombinant protein expression is more than 50% of the amount of cell proteins). With the selected strain are low-density fermentation in a 10-liter fermenter. After 3 hours of thermally induced cultivation, cells are untwisted, washed in phosphate-saline buffer solution and stored at -70°s to use. From 10 liters of culture receive 80 g of wet cells. These wet cells suspended in phosphate buffer, destroy in the high-pressure homogenizer and centrifuged. Select samples for SDS-PAGE. The results show that the desired protein is present on the track of sediment, with densely stained band at the position corresponding to molecular mass of 15.5 kDa, and any staining hardly observed at the position corresponding to the liquid above the sediment; this suggests that the KGD-Sak mainly exists in the form of inclusion bodies.

E. Isolation, solubilization and resaturate tel inclusion

After the destruction by compression of 80 g of cells engineered strain centrifuged at a speed of 10000 rpm./min and get 20 g of inclusion bodies. After washing in phosphate buffer (FB, 0.05 mol/l, pH=5,2) inclusion body was dissolved in a solution containing 0.1 mol/l FB with a pH of 5.2, mol/l guanidine hydrochloride, 0,5% β-mercaptoethanol, and incubated at room temperature until the solution becomes transparent. After ultracentrifugation at a speed of 30000 rpm./min the precipitate discarded, and the liquid above the sediment is diluted to re-naturalization in FB (0.1 mol/l, pH 5.0) and 0.5% β-mercaptoethanol.

f. Chromatography on columns of Sephadex G-10 and S-Sepharose FF

After concentration by ultrafiltration (molecular weight 1000, Millipore), the liquid above the precipitate is filtered through a column of Sephadex G-10. The filtrate on a column of S-Sepharose FF, pre-equilibrated 10 fold volume (volume gaskets) phosphate buffer (0.1 mol/l, pH 5.0). To control the flow rate and peak detection of the protein using a chromatograph (Waters). After loading the column was washed to baseline (no signal) phosphate buffer and elute the product with a solution of sodium chloride with a gradient of 0-1 mol/l Select erwerbende faction. The distribution of the desired protein analyzed by the method of SDS-PAGE and determine the concentration by the Bradford method (reagents purchased by the company Bio-Rad).

All operations chromatographic analysis are common for specialists in this field of technology.

g. Identification and purity determination of molecular mass

The sample analyzing method of SDS-PAGE in accordance with the Laboratory inform the DG molecular cloning. After staining of the gel bright blue Coomassie R-250 being scanned by Imagemaster VDS (firm Pharmacia)to determine the purity and molecular mass of the protein. Accordingly, it has been found that the purity is more than 95%, and a molecular weight equal to approximately 15.5 kD.

h. Determination of biological activity

To determine the biological activity have been analyzing the number of plaques on casein gel (A.G. Pipemo et al., J. Exp. Med. 48(1), 223-234 (1978)) and the method of chromogenic substrate (H.R.Lijnen et al., J. Biol. Chem., 266, 11826-11832 (1991)). The specific activity is about 90000-100000 HU/mg To determine units see O.Q.Tang et al., Drug Biotechnol (Chinese), 4(1), 1-4 (1997).

i. The definition of constants Km and Kcat for complexes Sak-plasmin and KGD-Sak-plasmin

Stand Sak or KGD-Sak (2 µmol/l) with 2 µmol/l plasminogen, respectively, in 0.1 mol/l phosphate buffer at pH 7.4 and temperature 37°C for 30 min, to form complexes with plasmin. Then select a catalytic amount (5 nmol/l) complex for the interactions within 0-10 min in the reaction systems listed below, in 0.1 mol/l FB at pH 7.4 and temperature 37°C, the optical density (405 nm) every 30 seconds concentrations of plasminogen are averaged according to the three tests.

The reaction with the system The final concentration
Sak-plasmin (KGD-Sak-plasmin)5 nmol/l
Chromogenic substrate S-23901 mmol/l
The plasminogen1-30 µmol/l

Activation of plasminogen complex KGD-Sak-plasmin is described by the equation of Michaelis-Menten (tables).

Table 1c.
Mapping constants of the enzymatic kinetics of plasminogen activation by complex KGD-Sak-plasmin and Sak-plasmin
Km, umol/lKcat, with-1Kcat/Km
Sak-plasmin6,511,060,16
KGD-Sak-plasmin14,101,460,10

j. Determination of ability to polymerization

As a control, use the Sak natural type. Samples dissolved in physiological solution. Tests carried out at two concentrations of protein (30 mg/ml (high) and 3 mg/ml (low). The solution was kept at room temperature. Samples taken every 24 h and analyzed by electrophoresis.

At both concentrations of the protein the ability to polymerization KGD-Sak was significantly lower than the Sak natural the IPA.

k. Sensitivity test on Guinea pigs

To test the sensitivity of the recombinant staphylokinase natural type and mutant KGD-Sak dissolved in sterile saline at a concentration of 2500 units/ml For each injection take the intact capsules to prepare a sterile manner fresh solutions. Healthy Guinea pigs (20) randomly distributed into two groups, 10 animals each. These Guinea pigs injected intraperitoneally injection of 0.15 mg/kg of r-Sak or KGD-Sak every other day for three times. The first and second intravenous exposure dose of 0.3 mg/kg performed respectively at 14 and 21 days. Two healthy Guinea pigs, which did not do the injection, injected with 0.3 mg/kg of the above samples and see a similar reaction to exclude pharmacological and pathological interference associated with the samples.

In the group with injection of r-Sak natural type: 8 Guinea pigs showed positive reaction of the class IV and 2 Guinea pigs showed positive reaction of the class II.

In the group with injection KGD-Sak: 2 Guinea pigs showed positive reaction of the class I and the other animals had no detectable reaction.

The reaction of the class I:a soft cough
R is the action class II: coughing, tremors
The reaction of the class III:strong tremors
The response class IV:convulsion, spasm, incontinence of urine and feces, shock fatal.

The content of antibodies in the serum of Guinea pigs immunized within 1-3 weeks, determined by the method of enzyme linked immunosorbent assay (ELISA), in which the antigens used wt-Sak and KGD-Sak, respectively. In the first week the number of antibodies against any antigen was little. During the second week the number of antibodies groups wt-Sak (n=10) increases to 1:800, while the number of antibodies groups KGD-Sak (n=10) is 1:200. In the third week, the number of antibodies groups wt-Sak is increased to 1:3200, while in the group KGD-Sak is increased to 1:400. Thus, the immunogenicity of KGD-Sak is significantly reduced compared with the immunogenicity of wt-Sak.

The above results indicate that the immunogenicity of KGD-Sak is significantly reduced compared with the immunogenicity of wt-Sak.

l. Analysis of inhibition of platelet aggregation

Slowly centrifuged (acceleration 150 g, 10 min) fresh blood with the addition of 1/10 volume of a solution (110 mmol/l) anticoagulant - citrate to obtain a plasma enriched with platelets (SWEAT). Add KGD-Sak to SWEAT until the final concentration of 2 mmol the/l and the mixture was incubated 2 min at 37° With continuous stirring. Then add the adenosine diphosphate (ADP) to a final concentration of 2 µmol/l as an inductor. The degree of platelet aggregation determine within 5 minutes dual unit of platelet aggregation (CHRONO-LOG560). As a control using samples of r-Sak natural type (2 µmol/l) and saline. Adenosine diphosphate buy the company Sigma, and other reagents of analytical purity produced in China.

Found that the degree of aggregation in the group KGD-Sak (3,8%±1,5%, n=3) significantly lower than aggregation in groups r-Sak (64%±4%, n=3) and saline solution (60%±3%, n=3). This suggests that the KGD-Sak has a strong potential in the inhibition of platelet aggregation caused by ADP (figure 3).

m. Thrombolytic analysis on animals

Thrombolytic analysis on animals carried out with KGD-Sak obtained in the present invention, checking that the KGD-Sak retains the same thrombolytic properties that Sak natural type. For the introduction of KGD-Sak dissolved in sterile saline, as described in section k of this example.

Treatment of thrombosis of the femoral artery of the experimental rabbits KGD-Sak

In each group processed KGD-Sak handled Sak natural type and in the control group without treatment, contained 6 animals. With the help of the arteriography shows as to the treatment of the middle segment of the femoral artery was not seen. When you re shooting in 60 minutes after intravenous injection of 0.1 mg/kg KGD-Sak femoral artery becomes completely filled with the restoration of the cycle of blood, which is consistent with the effect in the group Sak natural type, while in the control group, the femoral artery becomes full.

Treatment givemy experimental rabbits with KGD-Sak

In each group processed KGD-Sak handled Sak natural type and in the control group without treatment, contained 6 animals. After 4 h after intraocular injection of 10-20 µg KGD-Sak observed lysis of thrombus givemy, and red blood cells settle and form a boundary surface with aqueous fluids. Intraocular blood tumor is removed after 24 hours This is consistent with the effect in the group Sak natural type. However, in the control group giveme not significantly changed.

Thrombolytic therapy with KGD-Sak is safe and effective in the treatment of acute myocardial infarction in experimental dogs, caused by thrombosis of a coronary artery. In the experimental group, consisting of 6 animals, dogs received a dose of KGD-Sak (0.3 mg/kg body weight) via intravenous infusion; in the control group consisting of 6 animals, dogs who received saline, instead KGD-Sak, via intravenous infusion. Before and after entering a dose, do coronary arteriography. To enter the dose found that the left anterior descending branch of the coronary artery is empty or not completely filled in animals of both groups. When conducting arteriography after 30 minutes of treatment found that the left anterior descending branch again filled, and the animals in the experimental group survive. As for the control group, not completely filled arteries there are no noticeable changes, and animals die within a few hours.

Thrombolytic therapy with KGD-Sak is safe and effective in the treatment of acute cerebral infarction in experimental pigs caused by thrombosis of intracranial arteries. In the experimental group, consisting of 6 animals, pigs received a dose of KGD-Sak (0.2 mg/kg body weight) via intravenous infusion; in the control group consisting of 6 animals, pigs received saline instead of KGD-Sak, via intravenous infusion. Before and after entering a dose, do intracranial DSA arteriography. To enter the dose found that some of intracranial arteries are not completely filled in animals of both groups. When angiography after 30 minutes of treatment found that h is of the intracranial arteries again filled, and animals in the experimental group survive. As for the control group, not completely filled intracranial arteries there are no noticeable changes, and animals die a few days later.

Specialists in this field of art without further detailed description will be able to use this invention in the light of previous recommendations. Thus, it should be understood that the preferred specific embodiments of the above for the purpose of illustration and in no way limit the scope of the invention.

All sources cited in the description that you entered in its entirety, by reference. The essential characteristics of the present invention will become obvious to a person skilled in this technical field from the preceding description. Moreover, can be implemented in various modifications and improvements of this invention, without deviating from the essence of the present invention. It is assumed that such modifications and improvements are included in the scope of the attached claims.

1. Bifunctional recombinant derivative staphylokinase with thrombolytic activities and anticoagulative agent that is different from staphylokinase wild-type and the fact that one or more amino acid residues between amino acid residues 104 and 113 staphylokinase the wild-type replaced by other amino acids with the formation of the sequence RGD or KGD sequence, consequently its ability to polymerization, as well as cellular and humoral immunogenicity significantly reduced in comparison with staphylokinase wild-type or truncated with NH2-end variant that retains the activity of the full-size form.

2. Recombinant derivative staphylokinase or its variant according to claim 1, in which the substituted amino acids are located in the cow field GO.

3. Recombinant derivative staphylokinase or its variant according to claim 1 which includes the sequence RGD or KGD sequence obtained by substitution of one or two amino acid residues of staphylokinase wild-type.

4. Recombinant derivative staphylokinase or its variant according to claim 3, characterized in that the phenylalanine at position 111 (Phe 111) staphylokinase wild-type is replaced by aspartic acid (Asp 111), a rest of the sequence remains unchanged.

5. Recombinant derivative staphylokinase or its variant according to claim 4, characterized in that the phenylalanine (Phe 111) staphylokinase wild-type is replaced by aspartic acid (Asp 111), lysine at position 109 (Lys 109) is replaced by arginine (Arg 109), and the rest of the sequence remains unchanged.

6. Recombinant derivative staphylokinase or its variant according to claims 1-5, characterized by the amino acid consistently is thew, which essentially corresponds to the sequence selected from the group consisting of

(a) sequences provided in SEQ ID NO:1;

b) the sequence shown in SEQ ID NO:3; and

c) NH2-terminal truncation of SEQ ID NO:1 or SEQ ID NO:3.

7. Recombinant derivative staphylokinase according to claim 6, characterized by the amino acid sequence SEQ ID NO:1 or SEQ ID NO:3, which removed 1-16 amino acids with NH2-the end, and Ser at position 16 optionally substituted with Lys.

8. Recombinant derivative staphylokinase according to claim 6, characterized by the amino acid sequence SEQ ID NO:1 or SEQ ID NO:3, which removed 1-10 amino acids with NH2-end.

9. Recombinant derivative staphylokinase according to claim 6, characterized by the amino acid sequence SEQ ID NO:1 or SEQ ID NO:3, which removed 1-6 amino acids with NH2-end.

10. Recombinant derivative staphylokinase according to claim 6, characterized by the amino acid sequence SEQ ID NO:1.

11. Recombinant derivative staphylokinase according to claim 6, characterized by the amino acid sequence SEQ ID NO:3.

12. A method of obtaining a bifunctional recombinant derivative staphylokinase, comprising the following stages:

a) obtaining a DNA fragment containing the coding sequence of the biologically aktivkohlefilter;

b) implementation of in vitro site-specific mutagenesis of the DNA fragment to replace one or more of the codons of amino acid residues between amino acid residues 104 and 113 staphylokinase wild type codons for other amino acids for the formation encoded in the derived sequence RGD or KGD sequence;

c) cloning the mutated DNA fragment into a suitable vector;

d) transformation or transfection of a suitable host cell with the recombinant vector;

e) culturing the host cell under conditions suitable for expression of the DNA fragment; and

f) extraction and purification of the desired derivative staphylokinase.

13. The method according to item 12, in which site-specific mutagenesis is carried out by three rounds of amplification using polymerase chain reaction with plasmid pST-Sak as a matrix using direct primed, reverse primer, and a mutant primer.

14. The method according to item 12, which used the vector is a prokaryotic expression vector pLY-4.

15. The method according to item 12, which used a host cell is an E. coli K802.

16. The method according to item 12, in which the cells are the owners of cultivated at 30-42°and the pH value approximately 6-8 with a decrease in the rate of mixing with the increase of optical density(OD).

17. The method according to item 12, in which the final product is recombinant staphylokinase obtained by cell disruption in high pressure after fermentation, harvesting Taurus include centrifugation, followed by separation and purification of the two-stage method.

18. Isolated DNA, which is characterized by a nucleotide sequence that essentially corresponds to the nucleotide sequence that encodes a recombinant derivative staphylokinase according to any one of claims 1 to 11.

19. Isolated DNA according p characterized by nucleotide sequence represented in SEQ ID NO:2.

20. Isolated DNA according p characterized by nucleotide sequence represented in SEQ ID NO:4.

21. The recombinant vector for the expression of recombinant derivative staphylokinase containing an isolated DNA according to any one of p-20.

22. A method of obtaining a bifunctional recombinant derivative staphylokinase, comprising the following steps: culturing the host cell comprising an isolated DNA according to any one of p-20 or the recombinant vector according to item 21; culturing the host cell under conditions suitable for expression of the isolated DNA; and extraction and purification of the desired recombinant derivative staphylokinase.

23. Pharmaceutical composition suitable for treatment of the sludge is the prevention of thrombosis, includes a therapeutically effective amount of recombinant derivative staphylokinase according to any one of claims 1 to 11 and a pharmacologically acceptable carrier.

24. A method of treating or preventing thrombosis, which comprises the administration to a patient the pharmaceutical composition according to item 23.

25. The method according to paragraph 24, in which thrombosis is an arterial thrombosis, intraocular, it does, or seepage of blood.



 

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