WO2003008594A1

WO2003008594A1 - Construction of the plasmid for expressing the thrombus-targeting thrombolytic fusion protein

Info

Publication number: WO2003008594A1
Application number: PCT/CN2001/001301
Authority: WO
Inventors: Shuhan Sun; Hongli Yan; Ruiwen Chen; Dexter H. H. Chun
Original assignee: Gene-Life Biotechnology (Shanghai) Limited
Priority date: 2001-07-20
Filing date: 2001-09-03
Publication date: 2003-01-30
Also published as: CN1188522C; CN1398972A

Abstract

The application provides a fusion gene anx32-scuPA that is fused by one gene coding for annexin 32 and another coding for the site 144 to 411 of the amino acid sequence of the single chain urokinase. The length of the cyclic double strand thermal induced plasmid (pJM-anx32-scuPA) of the fusion gene is 6.8 Kb. Transforming the E. coli using the plasmid (pJM-anx32-scuPA) produces the engineering bacterium K802 of the application. The fusion gene of the application can express the fusion protein complexed by the annexin 32 and the site 144 to 411 of the amino acid sequence of the single chain urokinase. The fusion protein has the double function of anticoagulation and thrombolytics. It has the high efficiency in treating thrombolytic disorders and will not cause the side effect of hemorrhage.

Description

Thrombosis-targeting thrombolytic protein expression plasmid and construction technology thereof

The present invention relates to the field of medical biotechnology, and in particular to the preparation of a thrombus-targeting thrombolytic protein expression plasmid by a genetic engineering method. Background technique

Thrombosis, such as acute myocardial infarction (AMI) and venous thromboembolism, is a type of cardiovascular disease that seriously endangers human health and life. In western countries, deaths due to thrombosis already account for the largest proportion of total deaths in the population. In China, with the economic development and the aging of the population, the number of patients with thrombosis is increasing, and the demand for antithrombotic drugs is increasing. Thrombolytic drugs commonly used in clinical practice are mainly plasminogen activators, such as streptokinase (SK), urokinase (UK), single-chain urokinase (scu-PA), and tissue-type plasminogen activator (t -PA), etc. Although these drugs have strong thrombolytic effects, they all have many disadvantages: (1) bleeding: because these drugs not only activate plasminogen in fibrin clots, but also activate plasma Plasminogen increases plasmin activity in the blood paddle. Plasma plasmin hydrolyzes coagulation factor VII, etc., thereby reducing coagulation factors and causing bleeding. (2) The half-life in the body is short and the amount of treatment is large. (3) Re-infarction: The fibrinolytic system can activate platelets after activation, so that fibrinogen and Von Willebrand factor can accumulate on platelet GpIIb / IIIa receptors, and promote thrombosis. Therefore, thrombosis easily occurs again within a short time after thrombolysis, which makes the treatment fail.

Low-molecular-weight single-chain urokinase (scu-PA (144 ~ 411)) is a derivative of single-chain urokinase (scu-PA). It is composed of amino acid residues 144 ~ 411 of scu-PA. -PA is similar, with less bleeding side effects, but lacks specific affinity for fibrin, so the efficiency of thrombolysis is not high, and its clinical application is limited.

Therefore, a new type of thrombolytic agent with stronger potency, better thrombolytic specificity, and effective prevention of re-embolization is a hot topic of current research on antithrombotic drugs. Summary of the Invention

A new gene, an Annexin 32 (Anx 32) gene, cloned from the cysticercus cerevisiae cDNA library by the inventors (Sun Shuhan et al., Molecular cloning of cDNA encoding antigens for cysticercosis, Chinese Parasitology and Parasitology Journal of Insects, 1997, 15 (1): 15-20). The gene encodes a 347 amino acid protein with a molecular weight of 38KDa. In the presence of calcium ions, Annexin 32 can have high affinity, Specifically binds acidic phospholipids. The blood coagulation process can be divided into three stages: (1) Platelet activation stage. The asymmetry of the platelet phospholipid membrane changed, and the phosphatidylserine (PS), which was originally on the inner side of the plasma membrane, was exposed. (2) Platelet surface reaction stage. From factor X to the formation of thrombin, this series of reactions need to be performed on the surface of the platelet membrane. (3) Fibrin formation stage. Thrombin converts fibrinogen into fibrin. Since annexin 32 can bind membrane phospholipids that activate platelets with high affinity, it can competitively inhibit the activation of coagulation factors and play an anticoagulant role.

On this basis, the present inventors conducted a fusion expression study of annexin 32 and scu-PA (144 to 411), and completed the present invention.

The present invention provides a fusion gene anx32- _SC uPA, the fusion gene comprising a gene encoding annexin 32 shown in sequence 1 of the sequence listing and a gene encoding amino acids 144 to 411 of single-chain urokinase shown in sequence 3 Fusion.

The present invention also provides a thermally-inducible expression plasmid pJM-anx32-scuPA, which is a circular double-stranded chain of 6.8 Kb in length and cloned with a protein encoding annexin 32 and single-chain urokinase amino acids 144 to 411. Gene sequence and contains? ^! ^ Promoter.

The invention further provides an engineering bacterium K802 (pJM- anx32- scuPA), which is E. coli K802

JLF1, deposited at the China Type Culture Collection, with accession number CCTCC No. M201020 o The fusion gene anx32- _SC uPA of the present invention is prepared by the following steps: The gene and sequence encoding annexin 32 shown in sequence 1 The genes encoding single-stranded urokinase amino acids 144 to 411 shown in Fig. 3 were amplified by PCR respectively; then they were ligated by overlapping region extension.

The expression plasmid pJM_anx32-scuPA of the present invention is constructed by double-digesting the fusion gene anx32-scuPA with EcoR I and Sal I, and then ligating the vector pJM with EcoR I and Sal I.

The fusion expression plasmid of annexin 32 and scu-PA (144 ~ 411) constructed by the inventors can fuse and express annexin 32 and scu-PA (144 ~ 411), and the expression products can retain their respective biological activities, thereby A new thrombolytic drug with dual functions of anticoagulation and thrombolysis can be obtained. As mentioned above, existing thrombolytic drugs mainly dissolve thrombus by activating plasmin, and platelets are not sensitive to the action of plasmin, so these drugs have a poor therapeutic effect on platelet-rich thrombosis. In addition to its anticoagulant function, annexin 32 specifically binds to activated platelets, so it can specifically bind scu-PA (144 ~ 411) to the thrombus site, which can greatly improve the Therapeutic effect of platelet-containing arterial thrombosis.

In addition, the present invention uses a heat-inducible expression vector, which can reduce the cost during large-scale cultivation, and is conducive to large-scale production. Because the biological activities of Annexin 32 and single-chain urokinase are not affected by glycosylation Therefore, the present invention may use a prokaryotic expression system, and a yeast expression system or a mammalian cell expression system may also be used as required. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Schematic diagram of plasmid pJM- anx32- scuPA constructed by overlapping region extension method

Figure 2 Electrophoresis of plasmid p JM- anx32- scuPA

Of which: Lane 1: DNA Marker

Lane 2: anx32 gene amplified by PCR # 1

Lane 3: PCR # 2 amplified low-molecular-weight single-chain urokinase gene

Lane 4: Plasmid pJM- anx32_scuPA was double digested with EcoR I and Sal I Figure 3 Expression and purification of fusion protein ANX32-SCUPA

Of which: Lane 1: Uninduced bacterial protein

Lane 2: 1 hour after induction

Lane 3: bacterial protein 2 hours after induction

Lane 4: bacterial protein 3 hours after induction

Lane 5: Supernatant after bacterial sonication (almost no protein of interest)

Lane 6: Cells precipitate after sonication (with large amounts of protein of interest)

Lane 7: Purified protein of interest

Lane 8: Protein molecular weight standards

Lane 9: Western blot indicates that the purified protein is a fusion protein containing single-chain urokinase

The anx32 full-length gene-containing plasmid _P UC19-anx32 used in the present invention is preserved by our laboratory (see Sun Shuhan et al., Molecular cloning of cDNA encoding antigens for cysticercosis diagnosis, Chinese Journal of Parasitology and Parasites, 1997, 15 (1 ): 15-20); Low-molecular-weight single-chain urokinase (scu-PA (144 ~ 411)) Gene refers to the human urokinase cDNA sequence (this sequence can be obtained in the gene database (Genbank), sequence number: M15476), where Some amino acids have been changed to E. coli preferred codons, which were artificially synthesized by Shanghai Shenggong Company. For specific modification methods, please refer to the literature (Ma Zhong et al .: High expression of pro-urokinase gene in E. coli, biochemistry and Acta Biophysica Sinica, 1995, 27 (1): 17-22); the heat-inducible expression vector pJM is derived from the plasmid pJLA-503, and was gifted by Dr. M. kreg (see M. kreg et al. Inducible expression vectors incorporating the Escherichia coli ap ^e translational initiation region, Gene, 52 (1987) 279-283); the host strain used for plasmid amplification was DH10B, and the host strain used for protein expression was K802 (both strains were purchased from GIBC0). Example 1 Obtaining the fusion gene anx32-scu-PA (144 ~ 411)

As shown in Figure 1, the gene encoding annexin 32 and the gene encoding single-stranded urokinase amino acids 144 to 411 were amplified by PCR respectively; then, the fusion gene anx32-scu-PA (144 ~ 41 1). Specific steps are as follows:

(1) Amplify all the genes encoding Annexin 32 gene with primers a and b. The primer a is 5, TG GAATTC ATGGCCTACTGTCGCTCCC3 ', the primer is consistent with the 5 end of the anx32 coding sequence, and an EcoR I restriction enzyme site is introduced. Primer b is: 5 'GCCACACTGAAATTTTAA / TGCAGGGCCGATGAG3', in which the 18 'end of the 5' end is identical to the 5 end of the single-chain urokinase coding sequence, and the latter 15 bp is complementary to the 3 'end of the anx32 gene. High-fidelity pfu DNA polymerase was used for the PCR reaction. The concentration of dNTP was 20M, the concentration of Mg ²⁺ was 1.5 mM, and the denaturation, annealing, and extension temperatures were 94 ° C, 55 ° C, and 72 ° C, respectively, and the time was 45 seconds. , 4 seconds and 1 minute, a total of 30 cycles.

(2) The primers c and d were used to amplify a coding sequence fragment of a low molecular weight single-chain urokinase (scu-PA (144 to 411)). Primer c is: 5, CTCATCGGCCCTGCA / TTAAAATTTCAGTGTGGC3 '. The 5' end of the primer is 15 bp consistent with the 3 'end of anx32, and the latter 18 bp is consistent with the sequence of the 5' end of the low molecular weight single-chain urokinase. Primer d is: 5'TTGTCGAC GCA GAG GGC CAG GCC3, which is complementary to the 3 'end sequence of the low molecular weight single-chain urokinase coding sequence. A Sal I restriction enzyme site was also introduced. The PCR reaction procedure is the same as (1), and a total of 30 cycles are performed.

(3) A fusion gene encoding annexin 32 and a low-molecular-weight single-chain urokinase was obtained by the overlapping region extension method. The products obtained by the above two-step PCR reactions were separated by 1.0% agarose electrophoresis, and then recovered by gel. The recovered product was denatured at 94 ° C for 5 minutes, and the products of the two PCRs were mixed and annealed at 50 ° C. Because primers b and c have overlapping portions, the single strand of the anx32 gene can be paired with the single strand of scu-PA (14: ~ 411) after annealing. After adding DNA polymerase, MgCl ₂ , 10 X PCR buffer, and dNTP, PCR amplification was performed. Using this method, two genes were linked into one fusion gene anx32-scu-PA (144 ~ 411). In order to ensure high-fidelity amplification, the entire PCR program uses TaKaRa's pfu DNA polymerase, 50μ1 reaction system, denaturation, annealing, and extension temperatures of 94 ° C, 50 ° C, and 72 ° C, respectively, for 45 minutes. 20 seconds, 45 seconds and 1 minute Loop (for specific methods, see "Molecular Cloning Strategies for Targeting New Genes-Theory and Methods", Military Medical Science Press, 1999). Example 2 Construction of expression plasmid pJM-anx32-scuPA

The primers a and d were used to sequence the fusion gene anx32-scu-PA (144 ~ 411) amplified by the above method, and the sequencing reaction was completed by Shanghai Ji Kang Company. After the sequencing is correct, it is double-digested with EcoR I and Sai l and ligated to the vector pJM double-digested with EcoR I and Sal I. Restriction enzymes and T ₄ ligases for enzyme digestion and ligation reactions were purchased from TaKaRa Company, and the reaction was performed using the system recommended in the enzyme instructions. The heat-inducible expression vector pJM present invention utilized the full length 4. 9Kb (kilobase pairs), containing the phage P _R PL promoter, heat-inducible gene CI1857, plasmid origin of replication (ori) and ampicillin resistance gene (AP ^r ).

The specific reaction process is as follows: EcoR I and Sal I are used to double-digest the above PCR products and vectors. The reaction uses a high-salt (H) buffer system and 37 ° C for 3 hours. It was then separated by 1.0% agarose electrophoresis and recovered as a gel. The enzyme fragments and the carrier were mixed at a molar ratio of 5: 1, and the corresponding ligation buffer solution and ligase were added, and the ligation was performed at 16 ° C for 12 hours. Example 3 Preparation of Engineering Bacteria

The ligated product was transformed into E. coli strain K802 to obtain an engineered strain K802 (pJM-anx32-scuPA).

The specific steps are as follows: The host bacteria K802 is first cultured in 50ml LB medium (containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride, PH7.0) at 37 ° C with shaking to 0D. At 0.4 o'clock, centrifuge at 4000 rpm at 4 ° C to collect the host bacteria. After removing the supernatant, resuspend it in 1-2 ml of ice-cold 100 mmol / L calcium chloride solution to obtain the competent state. bacterial. 100 ng of the constructed plasmid and competent bacteria were cooled in an ice bath for 30 minutes, heat-shocked at 42 ° C for 90 seconds, and then cooled in an ice bath for 10 minutes. Add 0.8 ml of LB and incubate at 37 ° C for 1 hour, then take 0.2 ml of the transformation product and coat it on the LB agar plate containing the corresponding antibiotic, and incubate at 37 ° C for 12-15 hours to obtain a monoclonal Host bacteria containing recombinant plasmids. Extracting the plasmid of the host strain and cutting it with EcoR I and Sal I could cut out a band of 1.8 Kb. The host strain was a positive recombinant (Figure 2). The E. coli strain K802 containing the plasmid _P JM-anx32-scuPA is an engineered strain K802 (pJM-anx32_scuPA). Example 4 Expression and purification of fusion protein ANX32-SCUPA

See Figure 3, Engineering bacteria K802 (pJM- anx32- scuPA) can express the fusion protein ANX32-SCUPA (lane 2, 3, 4) of fusion protein Annexin 32 and low-molecular-weight single-chain urokinase by way of temperature induction. Most proteins are in the form of inclusion bodies (lanes 5, 6), and their expression can account for more than 20% of the bacterial protein. After protein purification, 10% SDS-PAGE electrophoresis and staining with Coomassie Brilliant Blue showed a single band with a molecular weight of 68 kDa (lane 7).

(1) Fusion protein induction expression

A single colony of the above-mentioned engineered bacteria was individually picked into 100 ml of LB medium containing 50 mg / L of ampicillin, and cultured at 28 ° overnight with shaking. On the next day, inoculate 100ml of bacterial solution into 1L LB medium containing 50mg / L of ampicillin, continue shaking culture at 28 ° C, until the bacteria grow to the logarithmic phase (detected by spectrophotometer, the absorbance at a wavelength of 600 dishes A _6QQ = 0. 4- 0. 5), the culture solution was quickly heated to 42 ° C in a 70 ° C hot water bath, and then cultured at 42 ° C with shaking for 5 to 7 hours. Finally, A _6QQ can be increased to 1. 2 or so.

(2) Isolation of inclusion bodies and preparation of crude protein

Centrifuged the bacterial solution at 7700g for 10 minutes to collect bacterial cells. Buffer solution (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8. 0) was added to the suspension at a rate of 3 ml per gram of wet cell pellet to suspend the pellet. Add lysozyme to a final concentration of 1 mg / mL, and ice-bath at 0 ° C for 15 minutes; then sonicate the bacteria, ultrasonic power 100 W, 30 seconds / time, and check the bacterial cells for breakage. Stop the ultrasound when the bacteria are basically broken. Centrifuge the sonication solution at 10,000 g for 15 minutes. Discard the supernatant. The pellet was washed twice with a 9-fold volume of washing solution (50 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, pH 8. 0).

The inclusion body prepared by the above method was added with 5 ml of lysate (8 M urea, 0.5 M NH ₄ C1, 500 mM Tris-HCl, pH 8. 5), and shaken at room temperature for 4 hours. The 7700 g was then centrifuged to remove the insoluble fraction. This method can be used to prepare about 70% crude protein.

(3) Purification of fusion protein

The crude protein was further purified by a Sephacryl S-400 gel chromatography column (Pharmacia). Equilibrium and eluent were 5 M urea, 0.5 NH ₄ C1, 50 mM Tris-HCl, pH 8. 0, 0.5 mM EDTA, 10 mM DTT o SDS-PAGE analysis, and collected the wash containing the protein of interest Decant, store at -20 ° C.

The sample was dialyzed for 12 hours. The dialysate was 10 times the volume of 5 M urea, 0.5 NH ₄ C1, 50 mM Tris-HCl, pH 8.5. Urokinase contains 12 pairs of disulfide bonds, so care should be taken to remove oxygen from the dialysate during dialysis. The dialysate was first degassed by ultrasound for 10 minutes, then passed through for 5 minutes, and finally the dialyzed Erlenmeyer flask was sealed with a parafilm membrane. The dialyzed sample was added dropwise to a 100-fold volume of buffer containing 2 M urea, 0.5 NH ₄ C1, 50 mM Tris-HCl, 0.5 mM EDTA, 1. 25 mM reduced glutathione, 0 5 mM oxidized glutathione, pH 8.5. Gently agitate at 4 ° C for 24 hours, and then add 0.5 mM oxidized glutathione, and continue agitating for 24 hours. Finally, the samples were dialyzed in a 12-fold volume of buffer solution 10 mM Tris-HC1, pH 8.0 for 12 hours. The dialyzed sample was concentrated using an ultrafiltration membrane.

The above samples were then subjected to ion exchange chromatography using a weak anion exchange column (the chromatography medium was diethylaminoethyl agarose, DEAE Sepharose Fast Flow, Pharmacia). The column was equilibrated with 50 mM Tris · HCl, pH 8.0. The eluted salt concentration was eluted at a concentration gradient from 0 to 1 mol / L NaCl at a flow rate of 1 ml / min. Polyacrylamide gel electrophoresis (SDS-P AGE) was used to determine the peak of the recombinant protein, and it was found that the recombinant protein was eluted at a salt concentration of 0.4 M. The peak eluate was collected and separated by 10% SDS-PAGE electrophoresis. Coomassie brilliant blue staining showed a single protein band of 68 kDa, indicating that a pure recombinant protein ANX32-SCUPA had been obtained.

The expression of recombinant protein was detected by Western blot. The specific method is: After the expressed recombinant protein is separated by 10% SDS-PAGE, it is electrically transferred to a nitrocellulose membrane with a current of 0.65 mA / cm ² and a time of 2 hours. Add TBST buffer (10 mM Tris-HC1, 150 mM NaCl, 0.05% Tween-20, pH 8.3) and wash three times. Add TBST + 5% skimmed milk powder and shake at 37 ° C for 1 hour. Then the blocking solution was discarded, and an antibody against single-chain urokinase was added at a ratio of 1:50, and shaken at 37 ° C for 1 hour. Discard the above liquid, wash with TBST three times, add secondary antibody, and shake at 37 ° C for 30 minutes. It was washed three times with TBST and developed by adding a diaminobenzidine coloring solution. The result was a single band (Figure 3, lane 9). For the above methods, please refer to "Modern Molecular Biology Experimental Technology" edited by Lu Shengdong, second edition, pages 400-403.

Coomassie brilliant blue staining (Bradford method) was used to determine the concentration of purified protein and the recovery was calculated. The resulting protein was freeze-dried and divided into aliquots and stored at -20 ° C.

Applicant or agent file number 0136S3 International application number PCT / CN 01/0 130 1 Notes on the preservation of microbiota

(Rule 13bis)

A. Explanation of the microorganisms described on page 2 of the specification, line -1 ^ _. .

B. Preservation items E. coli Other deposits are on the supplementary page □ Name of the depository China Type Culture Collection Center Address of the depository Wuhan University, Wuhan 430072, China

(Including postal code and country name)

D. This note is made for the following designated countries (if the note is not made for all designated countries)

E. Supplementary notes (if necessary).

The following description will then be provided to the International Bureau (type the description, for example: "deposited number")

"" To be filled in by the receiving Office

^ This page is already with the international application □ Date of receipt of this page by the International Bureau: Authorized Officer Authorized Officer

Form PCT / RO / 134 (July 1992):;

Claims

Claim

1. A fusion gene anx32-scuPA, said fusion gene comprising a gene encoding annexin and a sequence shown in sequence 1 A fusion of a gene encoding a single-chain urokinase amino acids 144 to 411 shown in sequence 3: Sequence 1:

1 atggcctact gtcgctccct ggttcatcta tatgccccca atggagagaa gtacaaaccg 61 actattaccc caacacccgg gttctcaccg accgctgatg ctgagcactt gaagcgtgca 121 atgcgaggac ttggcacgaa tgaacgtgcg atcattgaca ttcttggaaa ccgaacttca 181 gccgaaagaa tggccattcg tgacgcctat ccgtcgattt ccagcaagac cctgcacgat 241 gctctaacca gcgagctgag tggcaagttc cggaggttcg ccttgttgct aatccaatca 301 ccgtggcagg tgatggcaga ggctctttac gacgccatga agggggctgg cactaaggaa 361 cgcgtactca atgaaattat tgccgggtgt tcaaaggatg acatccctca gttgaaaaaa 421 gcttttgaag aagtgagcgg aggagaaacc cttgatgatg cgatcaaggg ggacacgagt 481 ggcgactacc gcgaggccct tctgctagcg ctcgccggtc aggctgatga accacaggcg 541 atgcaactca aaaacctaac accctccact ctcagtcagg ttgtgaatcc cggccttgct 601 gaaacggatg cgaaggagct gtacgcctgc ggtgaggggc gcccgggcac agcagagagt 661 cgtttcatgc gtcctatcgt caatcgctca ttccttcaat taaacgcaac gaatgaggct 721 tacaatcggg cctacggtca cccgttgatt gatgcaataa agaaggagac gtcgagagac 781 cttgaggact ttctcataac tagagttcgc tacgccactg atcgcgccag tctgtttgcc 841 gaactccttc actttgcc at gagaggagct ggcaccaagg actccacttt gcaacgtgtt 903

Sequence 3:

1 aagaattaaa atttcagtgt ggccaaaaga ctctgaggcc ccgctttaag attattgggg 61 gagaattcac caccatcgag aaccagccct ggtttgcggc catctacagg aggcaccggg 121 ggggctctgt cacctacgtg tgtggaggca gcctcatcag cccttgctgg gtgatcagcg 181 ccacacactg cttcattgat tacccaaaga aggaggacta catcgtctac ctgggtcgct 241 caaggcttaa ctccaacacg caaggggaga tgaagtttga ggtggaaaac ctcatcctac 301 acaaggacta cagcgctgac acgcttgctc accacaacga cattgccttg ctgaagatcc 361 gttccaagga gggcaggtgt gcgcagccat cccggactat acagaccatc tgcctgccct 401 atgtataaag cgatccccag tttggcacaa gctgtgagat cactggcttt ggaaaagaga 481 attctaccga ctatctctat ccggagcagc tgaaaatgac tgttgtgaag ctgatttccc 541 accgggagtg tcagcagccc cactactacg gctctgaagt caccaccaaa atgctatgtg 601 ctgctgaccc ccaatggaaa acagattcct gccagggaga ctcaggggga cccctcgtct 661 gttccctcca aggccgcatg actttgactg gaattgtgag ctggggccgt ggatgtgccc 721 tgaaggacaa gccaggcgtc tacacgagag tctcacactt cttaccctgg atccgcagtc 781 acaccaagga agagaatggc c.

2. A method for preparing a fusion gene an _X 32- _SCU PA, which comprises the following steps: The gene encoding annexin 32 is as follows:

1 atggcctact gtcgctccct ggttcatcta tatgccccca atggagagaa gtacaaaccg

61 actattaccc caacacccgg gttctcaccg accgctgatg ctgagcactt gaagcgtgca

121 atgcgaggac ttggcacgaa tgaacgtgcg atcattgaca ttcttggaaa ccgaacttca

181 gccgaaagaa tggccattcg tgacgcctat ccgtcgattt ccagcaagac cctgcacgat

241 gctctaacca gcgagctgag tggcaagttc cggaggttcg ccttgttgct as tccas tca

301 ccgtggcagg tgatggcaga ggctctttac gacgccatga agggggctgg cactaaggaa

361 cgcgtactca atgaaattat tgccgggtgt tcaaaggatg acatccctca gttgaaaaaa

421 gcttttgaag aagtgagcgg aggagaaacc cttgatgatg cgatcaaggg ggacacgagt

481 ggcgactacc gcgaggccct tctgctagcg ctcgccggtc aggctgatga accacaggcg

541 atgcaactca aaaacctaac accctccact ctcagtcagg ttgtgaatcc cggccttgct

601 gaaacggatg cgaaggagct gtacgcctgc ggtgaggggc gcccgggcac agcagagagt

661 cgtttcatgc gtcctatcgt caatcgctca ttccttcaat taaacgcaac gaatgaggct

721 tacaatcggg cctacggtca cccgttgatt gatgcaataa agaaggagac gtcgagagac

781 cttgaggact ttctcataac tagagttcgc tacgccactg atcgcgccag tctgtttgcc

841 gaactccttc actttgccat gagaggagct ggcaccaagg actccacttt gcaacgtgtt

901 cttgccttga gggctgatac tgatctagga agcatcaagg agaagtatgc ggagctctat

961 ggtgaaacct tggaagcggc aatcaagggt gatacttctg gtgactatga ggctctctgc

1021 ttgaaactca tcggccctgc ataa

And genes encoding single-chain urokinase amino acids 144-411

1 aagaattaaa atttcagtgt ggccaaaaga ctctgaggcc ccgctttaag attattgggg 61 gagaattcac caccatcgag aaccagccct ggtttgcggc catctacagg aggcaccggg 121 ggggctctgt cacctacgtg tgtggaggca gcctcacag cccttgctgg 181 ccacacactg cttcattgat tacccaaaga aggaggacta catcgtctac ctgggtcgct 241 caaggcttaa ctccaacacg caaggggaga tgaagtttga ggtggaaaac ctcatcctac 301 acaaggacta cagcgctgac acgcttgctc accacaacga cattgccttg ctgaagatcc 362gttccaagga gggcaggtgt gcgcagccat cccggactat acagaccatc tgcctgccct 402 atgtataaag cgatccccag tttggcacaa gctgtgagat cactggcttt ggaaaagaga

481 attctaccga ctatctctat ccggagcagc tgaaaatgac tgttgtgaag ctgatttccc 541 accgggagtg tcagcagccc cactactacg gctctgaagt caccaccaaa atgctatgtg 601 ctgctgaccc ccaatggaaa acagattcct gccagggaga ctcaggggga cccctcgtct 661 gttccctcca aggccgcatg actttgactg gaattgtgag ctggggccgt ggatgtgccc 721 tgaaggacaa gccaggcgtc tacacgagag tctcacactt cttaccctgg atccgcagtc

781 acaccaagga agagaatggc c

Amplified by PCR method respectively; then ligated by overlapping region extension method.

3. Expression plasmid pJM- anx32-scuPA, said expression plasmid is a circular double-stranded chain of 6.8 Kb in length, cloned with a gene sequence encoding annexin 32 and single-chain urokinase amino acids 144 to 411, and contains? !! ^ Promoter.

The plasmid expression vector according to claim 3, wherein the expression vector is heat-inducible.

5. A method for constructing an expression plasmid pJTM-anx32-scuPA, which comprises the following steps: The fusion gene anx32-scuPA is double-digested with EcoR I and Sal I, and then the vector PJM double-digested with EcoR I and Sal I is digested. connection.

6. Engineering bacteria K802 (pJM- anx32- scuPA), which is E. coli K802 and F1, is deposited in the China Type Culture Collection and its accession number is CCTCC No. M201020.