WO1995027048A1

WO1995027048A1 - A method of preparing recombinant staphylokinase

Info

Publication number: WO1995027048A1
Application number: PCT/CN1995/000025
Authority: WO
Inventors: Houyan Song
Original assignee: Shanghai Medical University
Priority date: 1994-04-04
Filing date: 1995-04-03
Publication date: 1995-10-12
Also published as: CN1096325A; CN1035192C

Abstract

The present invention relates to constructing expression vector pSTE-SAK-1 by using staphylokinase amplified by PCR and prokaryotic expression vector, such as PLY-4, and transforming E. coli with pSTE-SAK-1, and inducing the expression of such gene by heating. The engineered bacteria are propagated by fermenting, and destructed. The r-SAK product is purified from the supernatant by a two-step method which includes ion exchange and gel filtration; the purity and the yield of the staphylokinase of the invention are high, and cost low.

Description

Technical field of preparation of recombinant staphylokinase

The present invention relates to the field of biotechnology, and more particularly, to a method for preparing recombinant staphylokinase by using biotechnology. Background technique

Staphylokinase (SaK) is a proteolytic enzyme synthesized by lysogenic phage of Staphylococcus aureus. Staphylokinase has a molecular weight of about 15.5KD. The process of activating plasminogen (pig) is similar to streptokinase. First SAK and pig combine to form a 1: 1 complex, and then the complex activates another molecular pig to form Plasmin, which catalyzes the hydrolysis of fibrin, dissolves blood clots and restores blood circulation. In plasma, after SAK and pig form a complex SAK-plg, if no fibrin is present, the complex is quickly neutralized by α ₂ -antiplasmin and then phagocytosed by macrophages; in the presence of fibrin At this time, the lysine binding site on the pig molecule in the SAK-plg complex binds to fibrin and loses the binding site with α ₂ -antiplasmin, which is not inhibited by it, so it can not only activate the thrombin efficiently. Pigs specifically dissolve blood clots and are not prone to induce systemic fibrinolysis. The animal thrombolytic model also proved that SAK did not cause a significant decrease in fibrinogen while dissolving the thrombus. In addition, SAK has the following outstanding advantages: small molecular weight, good penetrability, dissolving effect on platelet-rich thrombus, especially cerebral arterial thrombus and limb arteriovenous thrombus, and has better curative effect.

However, since 1948, Lack CH et al. (Nature 1948; 161: 559-560) discovered that SAK synthesized by Staphylococcus aureus can activate plasma proteases for a long time, due to various reasons, such as Staphylococcus aureus synthesis and secretion of SAK levels Very low, it is difficult to remove impurities such as endotoxin when purifying SAK, so the research progress on the structure, function and clinical application value of SAK is slow. In recent years, with the development of recombinant DNA technology, Sako T. et al. (Mol- Gen- Genet 1983; 190 (2): 271- 177) isolated phage containing SAK gene from Staphylococcus aureus, and from the phage genome Cut out the SAK gene and Promoters and other regulatory sequences were recombined with the PBR322 vector to construct a clone containing the recombinant staphylokinase (r-SAK) gene and achieve expression in E. coli. Behnke D. et al. (Mol-Gen-Genet 1987; 210 (30): 528-534) used yeast to express the SAK gene. In 1993, Collen D. et al. (Fibrinolysis 1992; 6: 203-213) used a similar method of Sako T. to isolate the SAK gene and express it in E. coli after recombination with PUC19. The expression products existed in the culture solution, the surrounding membrane space and the bacteria. In vivo, pure <6mg / L culture was obtained after ion exchange, affinity chromatography and gel filtration. The methods of gene construction and product purification established by the above laboratories are complex and yields are low.

The object of the present invention is to provide a new method for preparing recombinant staphylokinase, which has a simple purification method, high product purity, high yield, and low cost. Brief description of the invention

The invention provides a method for preparing recombinant staphylokinase (r-S A K):

(1) Design primers based on the nucleotide sequence of the natural SAK gene of hemolytic Staphylococcus aureus, and use the SAK gene-containing Staphylococcus macromolecule DNA as a template to amplify the SAK gene by PCR;

(2) recombining the SAK gene with a prokaryotic expression vector to form an expression vector;

(3) transforming the host bacteria with an expression vector, and screening the transformed host bacteria;

(4) culturing the transformed host bacteria under appropriate conditions and inducing expression of the r-SAK gene;

(5) Isolate and purify the expressed r-SAK. Detailed description of the invention

As used herein, the term "primer" refers to a single-stranded DNA oligonucleotide sequence that can serve as a starting point for the synthesis of an extension that is partially or completely complementary to the nucleic acid strand to be amplified. The length and order of the primers must be such that they can initiate the synthesis of extension products. Preferably, the primers are about 5-50 nucleotides. The specific length and sequence depends on the desired target DNA or

The complexity of the RNA, and the conditions under which the primers are used, such as temperature and ionic strength.

The term "vector" may include a plasmid, a cosmid end plasmid, a phage, or a virus. Preferably, the vector is a prokaryotic expression plasmid. PCR amplification product cloned into the appropriate site of the expression vector Expression vector.

Once the expression vector is prepared, the expression vector can be introduced into an appropriate host cell by any of a series of suitable methods, such as by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, and the like.

The host cell used in the present invention may be a cell of a prokaryotic or eukaryotic organism. Ideal prokaryotic hosts include bacteria of the genus Escherichia coli, B. subtilis, and the like. The most ideal prokaryotic host is E. coli. The ideal eukaryotic host is yeast.

After the vector is introduced, the host cells are grown on a selection medium, and cells containing the expression vector are selected.

After the host cell containing the expression vector is selected, it can be cultured under conditions suitable for the growth of the host cell, and the expression of the staphylokinase gene can be induced under appropriate conditions. Preferably, the expression of staphylokinase is induced by heat.

The staphylokinase gene expression product can be isolated and purified by isolation and purification methods well known to those skilled in the art to obtain a purified expression product.

More specific technical solutions of the present invention are:

(1) Isolate Staphylococcus aureus from the human body, select the culture with the highest fibrinolytic activity, and purify the staphylokinase from the culture medium. Determine the 5 amino acid sequences at the N and C ends as a reference for designing primers. DNA was used as a template, and the staphylokinase gene was directly amplified by PCR. After recombination with the PUC19 plasmid vector, the gene structure was confirmed by nucleotide sequence analysis.

(2) The r- SAK gene is recombined with a prokaryotic expression vector (such as a vector containing PL, PR promoter, CIt857 gene, 5sRNA termination signal and other regulatory elements) to form an expression vector, transform E. coli, and select transformed E. coli (using As engineering bacteria), the expression of r-SAK gene is induced by temperature; the expression product r-SAK exists in the cytoplasm in a soluble and active state, accounting for more than 40% of the bacterial protein.

(3) Fermented cultured transformed E. coli, in which transformed E. coli is cultured with a low nutrient solution, such as pSTE-SAK-1, and fed with LB, glucose, and rare element solution before and after temperature induction. The temperature during fermentation is 30-42. 'C is appropriate, D0 = 50—80%, pH = 6—8, and the stirring speed decreases with increasing DO.

(4) The engineering bacteria are crushed with high pressure, and after centrifugation, they are removed by two steps of ion exchange and gel filtration. Purification of R-SAK:

After adding stabilizers such as human serum albumin, sodium glutamate, and phosphate to pure product r-SAK, the microporous membrane was filtered to sterilize and freeze-dried into a white powdery finished product. Brief description of the drawings

Figure 1 shows the construction of the expression vector PLY-4.

Figure 2 shows the construction of the expression plasmid pSTE-SAK. Examples

In order to further understand the present invention, the following examples are provided to explain the present invention more specifically. The invention should not be limited by these examples. Any modification made by those skilled in the art based on the disclosure of the present invention will fall within the scope of the claims of the present invention.

Example (1):

1. Isolate 50 strains of Staphylococcus aureus from human nasopharynx, inoculate them in LB culture medium and grow overnight, centrifuge to collect bacteria, and use supernatant to measure SAK activity with fibrin plate, 8 strains can secrete SAK, of which NO 0.4 The highest activity, the use of bovine plasminogen instead of human plasminogen in the fibrin plate for identification test, it was proved that 1 ^ 0.4 secretion is 3/8 1 :.

A large amount of No. 4 Staphylococcus aureus was cultured, and the 15KD protein was purified by ion exchange and molecular sieve. The 15KD protein was proved to have fibrinolytic activity by inverting fibrin plates and other methods. Sequence analysis of three amino acids, the results are shown in the sequence table, used to design PCR primers.

2. Amplify staphylokinase gene by PCR

No. 4 Staphylococcus aureus was cultured, and polymer DNA was prepared according to the method of Frederick M. Ananbel et al.

Pick a single colony and inoculate it in LB culture solution (37'C overnight culture), centrifuge at 5000 rpm for 10 minutes, suspend the bacteria in the buffer, add SDS and proteinase K, and incubate at 5 (TC for 4 hours with an equal volume of chloroform / isoamyl alcohol) Extraction, centrifugation and layering, the upper layer was extracted with an equal volume of phenol / chloroform / isoamyl alcohol, and the upper layer was added with 0.6 times the volume of isopropanol. Centrifuged the precipitate, washed twice with 75% alcohol, and dried. 0。 Dissolved in TE buffer, UV spectroscopic detection proved that A260 / A280 is greater than 2.0.

The results of N- and C-terminal amino acid sequence determination were used to design PCR primers. The ester method was synthesized by a DNA synthesizer, and was purified to amplify the staphylokinase gene. The 5 'end of each PCR primer pair contains a restriction enzyme recognition site.

Total PCR reaction volume ΙΟΟμΙ, containing:

Template DNA 0.5 μ _§

Primer I 50. 0 pmoles Primer I 50. 0 pmoles dNTP 2. 0 mmol / L

10 XPCR buffer

ddH ₂ 0

Taq DNA polymerase 2. 5U

During PCR reaction, 92 'C was denatured for 90 seconds, 70'C was extended for 60 seconds, and 52'C was annealed for 60 seconds. After 15 cycles, add 2.5 UTaq DNA polymerase, and perform another 15 cycles. After the reaction, take 2μ1 for agarose gel electrophoresis and detect with UV light. When using primers I and I for amplification: Primer I: 5'- "^ 'forward primer

5 'CGC GAA TTC ATG CTC AAA AGA AGT TTA -3'

Eco RI

Primer I: 3 '-^' reverse primer

5 'GCG GGA TCC TTT CTT TTC TAT AAC AAC CTT TGT -3'

Bam HI

A very bright specific band appears between 400 and 500 bp, and the length of the nucleic acid fragment contained in it matches the length of the complete staphylokinase gene.

3. Sequencing of staphylokinase genes

The gene amplified by PCR was recombined with the vector PUC-18 or 19 to form a recombinant plasmid. The nucleotide sequence of the staphylokinase gene was determined by DNA nucleotide analysis, and the amino acid sequence was derived from it.

4. Construction and identification of staphylokinase gene clones

After the PCR product is hydrolyzed with restriction enzymes, the PUC19 plasmid vector and T ₄ DNA ligase reaction system which have been completely hydrolyzed by the corresponding enzymes are added. After standing at room temperature for 1 hour, E. coli JM83 is transformed, and ampicillin-LB plates are used. Incubate in a 37 ° C incubator.

Pick a single colony to prepare a plasmid according to the Sambrook method, identify it by enzyme digestion, and present characteristics Sex strippers are named pST- SAK-1,

The nucleotide sequence and reading frame of the SAK gene in the plasmid pST-SAK-1 were confirmed by nucleotide sequence analysis.

5. Expression of staphylokinase gene clone in E. coli

4 Construction of Expression vector pLY- - in pGEM (3Z) LacZ gene LacZ removing portions of the carrier, and a solution containing Cits 857 genes and P _R? 1_ promoter and a multi-enzyme site, and 5sRNA termination signal and t ₂ stop codon, as shown in Figure 1.

Construction of expression plasmid—— After double enzymolysis of pST-SAK plasmid, it was separated by agarose gel electrophoresis, and the 400 ~ 500bp SAK gene was recovered, and the prokaryotic expression vector pLY-4 (containing PL, PR promoter, CIt857 gene, 55RNA termination signal and other regulatory elements) recombination, the reaction was transformed into Escherichia coli K802, screened and cultured with ampicillin-LB plate, as shown in Figure 2.

Pick a single colony, prepare a plasmid, and identify it by enzymatic hydrolysis. Those who contain a characteristic segment of the SAK gene are called the expression plasmid pSTE-SAK-1, and the engineering strain is pSTE-SAK-1.

Staphylokinase induced expression and identification of its expression products-pick single pSTE-SAK colonies, shake culture at 30'C in LB culture solution overnight, dilute with low-nutrition culture solution 1 to 100 the next day, and shake the bottle Medium 3CTC was cultured to A ₆ . . ~ 0.6 (about 4 ~ 5 hours), followed by induction culture for 3 hours, 5000r. Pm centrifugation to collect bacteria, washed twice with PBS (pH7.4), stored at 20'C or immediately separated for expression products .

6. Identification of expression products

SDS gel electrophoresis-performed according to Laemmli method.

Sample dissolving solution: 0.00625M Tris-HCl (pH 6.7), 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.001% bromophenol blue.

Sample processing and spotting: 1 ml of induction culture bacterial solution, after centrifugation, the pellet was suspended in 100 μl sample solution, and placed in a boiling water bath for 5 minutes to dissolve the pellet. Control bacteria or pre-induction bacteria were cultured in the same way and samples were treated under the same conditions. The spot size was 10 μl, and the amount of protein contained was almost equal.

Gel staining: Coomassie blue or silver staining.

Scanning of protein bands in the gel: Shimadzu CS-910 dual-wavelength scanner for transmission scanning, computer processing, and printing the percentage of each protein band. The bacterial lysate of induction culture had a very concentrated band at a molecular weight of 15KD, and the lysate or culture solution of the bacterial lysate before induction had almost no band at the same site.

Identification of the fibrinolytic activity of the expression product—the above gel was washed thoroughly with 2.5% Triton X-100 solution and distilled water in order, and covered on an agarose gel plate containing fibrinogen, human plasminogen and thrombin After 37'C incubation for 2 hours, a very clear translucent area appeared at 15KD, that is, the fibrin in this part had been dissolved, showing that the expressed product had fibrinolytic activity.

The state of existence of the expressed product in the bacterial body-After the bacteria were crushed with a high-pressure homogenizer, after ultracentrifugation at 35,000 rpm (about 10,000 g) for 60 minutes, the precipitate and supernatant were identified by SDS-PAGE and fibrinolytic activity, respectively. The lytically active band was distributed in the supernatant, and the precipitated part was hardly seen, indicating that the expression product Recombinant Staphylokinase (r-SAK) existed in the bacteria in a soluble and active state, and no inclusion bodies were formed. This feature is different from reports by D. Collen and others.

Detection of expression level—The Shimadzu CS-910 densitometer was used to scan the gel of SDS-PAGE Coomassie brilliant blue staining, and the results showed that r-SAK accounted for more than 40% of the total protein in the bacteria.

7. Determination of staphylokinase activity

The Lowry method was used to determine the protein content.

Determination of r-SAK activity-Given that there is no SAK activity unit and its standard, we use fibrin clot lysis method to determine r-SAK activity. And active states the following: In the reaction system lml, containing bovine fibrinogen 2mg, plasminogen 100m _g, 0. 4NIH units of thrombin, fibrin clot in an amount within 10 minutes complete dissolution of r- SAK It is 1HU.

Activity determination:

Preparation of standard curve (volume units are ul) Tube number 1 2 3 4 5 6 Standard r-SAK

(100u / ml) 100 80 80 40 20 0 human plasminogen

(lmg / ml) 100 100 100 100 100 100

PBS-0. 01% Tween 20

(pH7. 4) 590 610 630 650 670 690 Hook 37 'C 5min

Human thrombin 10 10 10 10 10 10 human fibrinogen

(lOmg / ml) 200 200 200 200 200 200 Shake 37'C insulation usually forms a clot after 1 to 2 minutes. Press the stopwatch. When the clot is completely dissolved, press the stopwatch. A stopwatch is required for each tube. The time (minutes) between the formation of the clot and the complete dissolution of the clot was recorded. The lOX lgT and lgSAK activity (U) were plotted.

Determination of samples:

measuring time (min) required for dissolution of the clot as described above after ^105-fold, to check the activity calculated from the standard curve and sample of SAK - lmg / ml diluted sample ^104.

Specific activity: r-SAK activity units per unit volume of solution / protein content per unit volume of solution (mg)

Example (2): Large-scale preparation of genetically engineered staphylokinase (r_SAK):

Engineering bacteria fermentation-

Shake flask fermentations: pSTE- SAK- 1 a single colony was inoculated LB- Amp liquid culture, 30Ό cultured overnight, the next day with basal medium 1: 100 dilution, with shaking sufficient to A _6. .

0.6-0.8 After induction culture for 3-4 hours, the bacterial solution was centrifuged with a known empty weight, 5000r. P. M., 4. Centrifuge at C for 10 minutes, discard the supernatant, wash the pellet with PBS (pH 7.4) once, weigh, and calculate the weight of the pellet.

5L NBS Bio Fill tank fermentation: pSTE — SAK — 1 single colony in 100ml LB monoculture solution 3CTC culture solution, test A ₆ . . As a seed liquid for fermentation.

Change the basic culture solution in the fermenter, set the temperature 30 ° C, pH 6-8, dissolved oxygen 50-80%, stirring speed 600 times / minute (the stirring speed varies with the oxygen content), and the parameters are stable. Then, inoculate the pSTE-SAK-1 bacterial solution at a volume ratio of 1: 100 (bacterial seed solution: culture solution). A ₆ . . When it reaches about 1.0, fermentation is induced for 3-4 hours while maintaining the above parameters.

The bacteria collection method is the same as that of shake flask culture: the bacteria can be stored at a 20'C for later use.

Purification of r-SAK——.

Broken bacteria

The wet bacteria were washed three times with 1000 ml of PBS (pH 7.4), suspended in PBS (10 _gm / 100 ml), squeezed with a high-pressure homogenizer pump, and centrifuged at 15000 r. Pm for 10 minutes to collect the supernatant.

Ion exchange

The S-Sepharose column was equilibrated with a buffer solution, the above sample was added, and the hybrid protein (3 times the bed volume) was eluted with the buffer solution. The pH gradient and the salt gradient buffer were used to elute r-SAK. Filter and concentrate.

Gel filtration

The gel column was equilibrated with PBS pH 7.4, and the ultrafiltration concentrated solution was eluted with PBS pH 7.4 to collect and combine each tube solution containing SAK activity. After filtering through 0.22μ microporous filter membrane, it was aseptically packed and lyophilized into a semi-finished product. After filtering through a microporous filter with a thickness of 0.22 _μm , and adding stabilizers such as human serum albumin, it was aseptically packaged and lyophilized into a finished product.

After crushing 68g of wet bacteria, the total protein measured by the Lowry method was 8.21g, and the total activity measured by the fibrin clot dissolution method was 2.% X 10 ⁸ HU, and the specific activity was 0.36 X 10 ⁵ HU / mg. The total protein of the supernatant of the press was 7.16g, the total activity was 2.93 X 10 ⁸ HU, and the specific activity was

0. 41 X 10 ⁵ HU / mg.

R-SAK crude product was separated by S-Sepharose to obtain r-SAK 2.0 g, active

1. 8 X 10 ⁸ HU. Specific activity 0.9 X 10 ⁵ HU / mg.

After the above r-SAK sample was desalted by gel filtration, r-SAK 1.6 g was obtained, the activity was 1.6 X 18 ⁸ HU, the specific activity was 10 ⁵ HU / mg, and the purity of r-SAK was 99%. Possibility of industrial application

The SAK gene and its regulatory sequence are reported from the phage genome as reported in the off-picture documents or patent documents. The present invention directly uses S. aureus macromolecular DNA as a template to amplify and prepare the SAK gene by PCR. The SAK gene constructed by the present invention uses a large intestine rod The bacteria efficiently express r-SAK, and r-SAK is soluble and active in the bacteria. The purification method is simple and the yield of the product is high. 400-500mg of pure product can be obtained per liter of fermentation broth, and the purity is 97-99%. Compared with the yield of 6 mg per liter of fermentation solution by D. Collen et al., The effect of the present invention is significantly improved, and its cost is much lower.

Table 1 lists the protein content, activity, and specific activity during each purification step.

Table 1 Protein content Total active iSi purification times

(g) (X 10 ⁸ HU) (X 10 ⁵ HU / mg)

Whole bacteria (68g wet bacteria) 8. 21 2. 96 0. 36

Supernatant 7. 1 2. 93 0. 41 1.2

S— Sepharose 2. 0 1. 8 0. 9 2. 5

Gel filtration 1. 89 1. 6 1. 0 2. 78

The r-SAK prepared by applying the invention has been proved by animal experiments to have good thrombolytic function, such as:

(1) r-SAK was used to treat experimental femoral arterial thrombosis in dogs, and angiography showed that the femoral artery was not developed below the treatment before the treatment. R-SAK 0.1 mg / kg was injected intravenously, and the femoral artery was fully filled after 60 minutes. Circulation was restored, and no femoral artery filling was seen in control animals. A total of 8 dogs were made (5 in the treatment group and 3 in the control group).

(2) The therapeutic effect of r-SAK on anterior chamber exudation in experimental rabbits: r-SAK10-20 _F g was injected intraocularly, and the exudate disappeared after 2-4 hours. The control group showed almost no change within 48 hours. There were 6 eyes in the experimental group and 4 eyes in the control group.

(3) Effect of r-SAK on experimental rabbits with anterior chamber hemorrhage ^: Intraocular injection of r-SAK 10-20 g, 4 hours later, the anterior chamber blood clot was dissolved, and the red blood cells formed an interface with the aqueous humor By 24 hours, the blood in the eye had been cleared. The hemorrhage in the anterior chamber of the control rabbit eyes was not significantly changed. There were 6 eyes in the experimental group and 4 eyes in the control group.

The finished recombinant staphylokinase prepared by the present invention is dissolved in water for injection and used for injection to treat myocardial infarction, pulmonary, brain, peripheral vascular thromboembolism, hemodialysis pathway and blood clotting in blood GTT AT GAAA AAG AAAAA T

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Claims

Claim

1. A method for preparing recombinant staphylokinase (r-SAK), which is characterized by:

(1) Design primers based on the nucleotide sequence of the natural SAK gene of hemolytic Staphylococcus aureus, and use the macromolecular DNA of the Staphylococcus aureus strain containing the SAK gene as a template to amplify the SAK gene by PCR;

(2) The SAK gene is recombined with the prokaryotic expression vector to form an expression vector.

) Culturing the transformed host bacteria under appropriate conditions, and inducing expression of the r-SAK gene;

(5) Isolate and purify the expressed r-SAK.

2. The method according to claim 1, wherein the hemolytic Staphylococcus aureus is a culture with the highest fibrinolytic activity isolated from the human body.

3. The method according to claim 1, wherein the primers are designed according to the 3 'and 5' ends of the sequence shown in the sequence table, and the preferred primers are:

5 '-CGC GAA TTC ATG CTC AAA AGA AGT TTA-3'

Eco RI

with

5 '-GCG GGA TCC TTT CTT TTC TAT AAC AAC CTT TGT-3'.

Bam HI

4. The method according to claim 1, wherein the prokaryotic expression vector contains a PLY-4 plasmid containing PL, PR promoter, CIts857 gene, 5sRNA termination signal and other regulatory elements.

5. The method of claim 1, wherein the host bacteria is E. coli.

6. The method of claim 1, wherein the expression of the SAK gene is induced by temperature.

7. The method according to claim 1, wherein the engineering bacteria transformed with the expression plasmid are further cultured on a large scale by a fermentation method.

8. The method according to claim 1, wherein the separation and purification of r-SAK is performed by ion exchange and gel filtration.

9. The method according to claim 1, further comprising adding a stabilizer to the purified r-SAK, filtering and sterilizing, and packaging and lyophilizing.