CN108517009A - Sj13 polypeptides and its application in preparing antithrombotic reagent - Google Patents

Abstract

The invention discloses a kind of Sj13 polypeptides and its mutant from Schistosoma japonicum to obtain recombinant polypeptide Sj13 and its mutant using technique for gene engineering；Through APTT, the detection of PT and TT coagulation functions, identifying Sj13 and its mutant has preferable external anticoagulating activity；It tests and detects through enzyme kinetics, it is found that Sj13 polypeptides and its mutant have the function of inhibiting blood coagulation correlation factor XIa, Xa and Plasmin；Through mouse FeCl₃Common carotid artery thrombus model is damaged, it is found that Sj13 inhibits carotid thrombus to be formed, there is anti-thrombus function, and bleeding risk is relatively low.Sj13 polypeptides and its mutant have important anticoagulation/antithrombotic reagent development and application value.

Description

Sj13 polypeptide and its application in the preparation of antithrombotic drugs

technical field

The invention belongs to the fields of genetic engineering and biomedicine, and specifically relates to Sj13 polypeptide derived from Schistosoma japonicum and its mutant, and its application in the preparation of anticoagulant and antithrombotic drugs.

Background technique

Thrombosis can cause ischemia, hypoxia, necrosis (arterial thrombosis) and congestion, edema (venous thrombosis) of corresponding tissues and organs, which seriously threaten people's life and health. Arterial thrombosis is the main cause of more than 90% of myocardial infarction and 80% of cerebral apoplexy, and arterial thromboembolic cardiovascular and cerebrovascular diseases are the leading cause of human death. Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the third leading cause of death from cardiovascular-related diseases after myocardial infarction and stroke. There are millions of VTE patients diagnosed every year in the world. In the United States, according to incomplete statistics, more than 900,000 cases of VTE occur each year, and about 300,000 of them die of pulmonary embolism. In Europe, there are 1.12 million cases of VTE and 543,000 cases of PTE every year. It is one of the main causes of VTE-related sudden death, accounting for about 5%-10% of hospital deaths. In my country, from 1997 to 2008, the statistical data of more than 60 tertiary hospitals in 24 regions for PTE showed that the incidence of PE in hospitalized patients increased year by year, and it was about 0.1% in 2008. In addition, from 2006 to 2010, 205 hospitals in 28 provinces and autonomous regions targeted specific groups of people, including intensive care unit (ICU), chronic obstructive pulmonary disease (COPD), lung cancer, elderly inpatients in internal medicine, and patients undergoing major obstetrics and gynecology surgery. According to the epidemiological survey of VTE, the incidence rates were 27%, 9.7%, 11.5%, and 9.7%. It can be seen that in the Chinese population, the incidence of VTE is even comparable to that in Europe and the United States, and the disease is seriously underestimated in China and even in Asia. Therefore, abnormal blood coagulation leads to embolism disease, which is the main killer of human health.

Anticoagulant drugs, as mainstream drugs for the prevention and treatment of venous thrombosis, can be traced back to the use of traditional anticoagulant drugs heparin and warfarin, with a history of more than 70 years.

In 1955, F.Markward discovered that European medical leeches had anticoagulant properties, and named them hirudin. Due to the serious bleeding side effects of hirudin, on December 15, 2000, Medicines, approved by the FDA, listed its researched and developed hirudin derivatives in the United States, Bivalirudin (Bivalirudin), which has 20 peptides synthesized, and blood coagulation Enzyme binding is reversible. Subsequently, AstraZeneca developed the first directly oral thrombin inhibitor, which is a 2-peptide similar to fibrinopeptide A, which can rapidly, reversibly and competitively bind to thrombin. However, the company withdrew the drug from the market worldwide in February 2006 because severe liver damage could occur after taking it. However, dabigatran etexilate, a new oral anticoagulant that directly inhibits thrombin developed by Boehringer Ingelheim in Germany in April 2008, was marketed in Germany and the United Kingdom, and it was approved by the FDA in the United States in October 2010. After warfarin, the first new oral anticoagulant.

In 1987, Tuszynski et al. isolated the first FX inhibitor from the salivary gland of the Mexican leech. In 1990, Waxman et al. isolated another FX inhibitor tick peptide (TPA) from the salivary gland of the African leech tick. In 1998, Bayer Company Started the FXa inhibitor research and development project, referring to the structure of tick peptide (TPA), a total of 200,000 compounds were screened out, and 13 lead compounds were selected from them. Rivaroxaban (rivaroxaban) was discovered in 1999. In September and October 2008, it was approved to increase sales in Canada and the European Union, and it is used for elective hip and knee replacement and prevention of VTE in adults. On March 31, 2009, it was approved by the China Food and Drug Administration (SFDA) and marketed in my country for the prevention of elective hip and knee replacement and VTE in adults. Rivaroxaban is the world's first oral direct FXa inhibitor, which can inhibit free and bound FXa with high selectivity. Since then, Pfizer and Bristol-Myers Squibb have jointly developed Apixaban (apixaban), which was launched in the European Union and the United States in May 2011 and December 2012 respectively, and was approved for sale in my country in 2013. Japan Sankyo Co., Ltd. developed Edoxaban (Edoxaban), which was launched in Japan and my country respectively in 2011.

Previous studies have found that there are active substances with anticoagulant function in human parasitic schistosomiasis. Among them, the research on the anticoagulant active substances in Schistosoma mansoni, which is widely prevalent in Africa, South America and Asia, is relatively clear. So far, SmAP, SmPDE, sK1, SmSP, Sm22.6, SHW4-2 and annexin have been found. Anticoagulant proteins, and anticoagulant small molecules such as eicosanoids, but no reports of anticoagulant polypeptides. In 2015, SL Ranasinghe et al. reported the first schistosome anticoagulant polypeptide SjKI-1, which comes from Schistosoma japonicum. SjKI-1 at 7.5 μM can prolong the partial thromboplastin time (APTT) by 2 times and has weak anticoagulant activity , there is no report about the follow-up study of this polypeptide.

Contents of the invention

The present invention obtains the Schistosoma protein sequence from the Schistosoma protein database through structural biology and bioinformatics; uses genetic engineering technology to clone, express and purify the Schistosoma japonicum polypeptide, and based on the APTT, PT and TT screening platforms, systematically Evaluated the anticoagulant function of Schistosoma japonicum polypeptide resources; tested the anticoagulant effect and bleeding risk of the screened polypeptide Sj13 through animal experiments; carried out the detection of coagulation factors and enzyme kinetics experiments by substrate chromogenic method, and revealed the anticoagulant effect of Sj13. coagulation mechanism; polypeptide site-directed mutagenesis method to explore the structure-activity relationship of anticoagulant polypeptide Sj13. The invention confirms that the polypeptide Sj13 is a natural lead drug molecule of a novel anticoagulant, and provides a new anticoagulant polypeptide resource for the preparation of novel anticoagulant and antithrombotic drugs.

The object of the invention is achieved through the following technical solutions:

The present invention provides a polypeptide Sj13, which comprises the amino acid sequence shown in SEO ID NO.1.

The present invention provides an isolated nucleic acid molecule encoding the polypeptide Sj13. Further, the nucleic acid molecule comprises the nucleotide sequence shown in SEO ID NO.2.

The present invention provides a polypeptide Sj13 mutant, the mutant has amino acid substitutions or substitutions at the P1 and P2' sites of the Sj13 polypeptide, the P1 site corresponds to R17 of SEQ ID NO.1, and the P2 The 'site corresponds to Y19 of SEQ ID NO.1; the replacement or substitution of the P1 site is preferably a basic amino acid residue, and the replacement or substitution of the P2' site is preferably a non-polar hydrophobic amino acid residue. Further, the above polypeptide Sj13 mutant comprises the amino acid sequence shown in SEO ID NO.9 or 10.

The present invention provides an isolated nucleic acid molecule encoding the polypeptide Sj13 mutant.

The present invention provides a vector, which comprises a nucleic acid molecule encoding the polypeptide Sj13, and/or a nucleic acid molecule encoding a mutant of the polypeptide Sj13.

The present invention provides a non-human cell, the cell comprising the polypeptide Sj13 and/or the nucleic acid molecule encoding the polypeptide Sj13; and/or the polypeptide Sj13 mutant and/or the nucleic acid molecule encoding the polypeptide Sj13 mutant.

The present invention provides a set of primers, the primer set includes 3 pairs of primers, respectively:

Sj13-FP1 shown in SEO ID NO.3 and Sj13-RP1 shown in SEO ID NO.4;

Sj13-FP2 shown in SEO ID NO.5 and Sj13-RP2 shown in SEO ID NO.6;

Sj13-FP3 shown in SEO ID NO.7 and Sj13-RP3 shown in SEO ID NO.8.

Further, the present invention also provides the use of the above primers in amplifying the nucleic acid molecule encoding the polypeptide Sj13.

The present invention provides a polypeptide Sj13, and/or the use of a nucleic acid molecule encoding the polypeptide Sj13 in the preparation of anticoagulant and antithrombotic preparations. The preparation can be selected from a kit, a drug, a pharmaceutical composition, etc., preferably a drug or a pharmaceutical composition.

On this basis, the present invention also provides a polypeptide Sj13 mutant, and/or the use of a nucleic acid molecule encoding the polypeptide Sj13 mutant in the preparation of anticoagulant and antithrombotic preparations. The preparation can be selected from a kit, a drug, a pharmaceutical composition, etc., preferably a drug or a pharmaceutical composition.

The present invention provides the use of a Sj13 polypeptide and its mutants in inhibiting coagulation-related factors XIa, Xa and/or Plasmin. Said use may denote both diagnostic or therapeutic and non-diagnostic or therapeutic purposes.

The beneficial effects of the present invention are:

1. Polypeptide Sj13 can significantly prolong the detection time of APTT; the common carotid artery thrombosis model was injured by 10% FeCl ₃ in C57BL/6 mice, and it was further found that polypeptide Sj13 can inhibit thrombus formation in animals; the mouse tail docking model evaluates the risk of bleeding is low . The anticoagulant activity of the polypeptide Sj13 is superior to that of the prior art SjKI-1, and has important application value for the development and application of anticoagulant/antithrombotic drugs.

2. Sj13 can inhibit FⅪa and FXa, but has no obvious effect on FIIa. Through the enzyme kinetics research of FⅪa and FXa, it is concluded that Sj13 is a mixed inhibitor of FⅪa and an anti-competitive inhibitor of FXa, suggesting that the Sj13 polypeptide is not a competitive inhibitor, and it mainly binds to the active center of FⅪa and FXa. area exerts an inhibitory effect.

3. Through site-directed mutagenesis experiments, the R and Y at the P1 and P2′ sites of the Sj13 polypeptide were mutated into A, respectively, and it was found that the mutant polypeptide Sj13Y19A had enhanced APTT prolongation and FⅪa activity, especially APTT enhancement of 17.8 Times, has important anticoagulant/antithrombotic drug development and application value.

Description of drawings

Figure 1. Plasmid map of prokaryotic expression system pET-28a(+).

Figure 2. Sj13 high performance liquid chromatography detection peak diagram: X-axis, program running time set by HPLC; Y-axis, absorbance value; wavelength is set to 270nm.

Figure 3. Sj13MOLDI-TOF-MS mass spectrometry detection diagram: X-axis, the mass-to-charge ratio (m/z) value of the substance; Y-axis, the relative intensity of the ion current; the arrow indicates the molecular weight of the recombinant Schistosoma protein.

Figure 4. Sj13 activated partial thrombin time (APTT) detection results: X-axis, the final concentration of recombinant schistosome protein, respectively 0 μg/ml, 1.04 μg/ml, 2.08 μg/ml, 4.17 μg/ml, 8.33 μg/ml , 16.67 μg/ml, 33.33 μg/ml; Y axis, APTT detection time.

Figure 5. Sj13 prothrombin time (PT) detection results: X-axis, the final concentration of recombinant schistosome protein, respectively 0μg/ml, 1.04μg/ml, 2.08μg/ml, 4.17μg/ml, 8.33μg/ml, 16.67μg/ml, 33.33μg/ml; Y axis, PT detection time.

Figure 6. Sj13 plasma thrombin time (TT) test results: X-axis, the final concentration of recombinant schistosome protein, respectively 0μg/ml, 1.04μg/ml, 2.08μg/ml, 4.17μg/ml, 8.33μg/ml, 16.67μg/ml, 33.33μg/ml; Y axis, TT detection time.

Figure 7. Functional verification of Sj13 on the common carotid artery thrombosis model of FeCl3 injury in C57BL/6 mice.

Figure 8. Effects of different treatments on the thrombosis of common carotid artery in mice injured by FeCl3: a. normal saline; b. heparin; c. low concentration of Sj13; d. high concentration of Sj13.

Figure 9. The effect of Sj13 on tail docking bleeding in mice.

Figure 10. The inhibitory effect of Sj13 on coagulation factors: X-axis, different concentrations of rSj13 (μM); Y-axis, residual enzyme activity of coagulation factors, 0 μM rSj13 residual enzyme activity is 100%. A: FⅫa final concentration: 0.2 (μg/ml), S2302: 800 μm. B: Final concentration of FⅪa: 0.78 (μg/ml), final concentration of S2366: 600 μM, IC50 value: 63 nM C: final concentration of FXa: 0.075 (μg/ml), final concentration of S2222: 600 μM, IC50 value: 3.327 nM. D: Final concentration of FIIa: 0.3125 (μg/ml), final concentration of S2238: 800 μM. E: Plasmine final concentration: 20nM, S2251 final concentration: 1mM, IC50 value: 78.125nM. F: Inhibitory effect of Sj13 (2.5 μM) on FⅫa, FⅪa, FXa, FIIa and Plasmine.

Figure 11. Kinetic detection results of Sj13 inhibiting FⅪa enzyme: A: Sj13, 1/V versus 1/[S] double reciprocal plot. The X-axis represents the reciprocal of different concentrations of S2236, and the Y-axis represents the reciprocal of the speed of Sj13 inhibiting the binding reaction between FⅪa and S2236 at the four concentrations. Different final concentrations of Sj13 assay; OnM, 19.53nM, 78.125nM, 156.25nM. Final concentrations of S2366; 0.42 mM, 0.83 mM, 1.67 mM. FⅪa final concentration: 0.75 (μg/ml). Regression equation: Sj13(0nM); y=36.426x+23.879, R ² =0.9997Sj13(19.53nM); y=39.69x+33.902, R ² =0.99; Sj13(78.125nM); y=43.708x+58.753, R ² =1; Sj13 (156.25nM); y=50.039x+77.755, R ² =0.9493. B: Km/Vmax plotted against different concentrations of Sj13, regression equation: y=0.0109x+18.547, R ² =0.9513. C; 1/Vmax was plotted against different concentrations of Sj13, the regression equation: y=0.4412x+24.481, R ² =0.9984.

Figure 12. Kinetic detection results of Sj13 inhibiting FXa enzyme: A: Sj13, 1/V versus 1/[S] double reciprocal plot. The X-axis represents the reciprocal of different concentrations of S2222, and the Y-axis represents the reciprocal of the speed of Sj13 inhibiting the binding reaction between FXa and S2236 at the four concentrations. Different final concentrations of Sj13 assay; OnM, 0.92nM, 2.44nM, 4.88nM. Different final concentrations of S2222 assay; 0.15mM, 0.3mM, 0.6mM. FXa final concentration: 0.075 (μg/ml). Regression equation: Sj13(0nM); y=9.3139x+41.623, R ² =0.9711; Sj13(0.92nM); y=8.8745x+54.293, R ² =0.9996; Sj13(2.44nM); y=7.018x+83.625 , R ² =0.9036; Sj13 (4.88nM); y=7.727x+117.26, R ² =0.9906. B: Km/Vmax plotted against different concentrations of Sj13, regression equation: y=15.734x+41.79, R ² =0.9949.

Figure 13. Effects of digestive enzymes on Sj13: X-axis: different concentrations of Sj13, final concentrations: 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM; Y-axis: residual enzyme activity of digestive enzymes. The IC50 value of trypsin (Trypsin) is 233×10-3M, and the IC50 value of chymotrypsin (α-Chymotrypsin) is 48×10-3M.

Figure 14. Structural diagram of polypeptide BPT and Sj13 protein.

Figure 15. Sj13 protein mutation site.

Fig. 16. Three-dimensional structural diagram of Sj13 protein and its mutant proteins.

Fig. 17. HPLC detection peak diagram of recombinant Sj13 mutant: X-axis: program run time set by high-performance liquid chromatography, Y-axis: absorbance value, wavelength set to 270nm. A and B respectively show the peak diagram of the protein elution peak separated from the Sj13 mutant protein concentrate by C18 reverse high performance liquid chromatography (RP-HPLC), and the peak indicated by the arrow is the collected protein elution peak . The time for Sj13R17A to collect protein solution: 21.94 minutes, and the time for Sj13Y1719A to collect protein solution: 20.786 minutes.

Figure 18. MOLDI-TOF-MS mass spectrometry detection graph of recombinant Sj13 mutant: X-axis: mass-to-charge ratio (m/z) value of the substance, Y-axis: relative intensity of ion current. Blue arrows indicate the molecular weight of the recombinant Sj13 mutant. A: mass spectrometry peak pattern of recombinant Sj13R17A, B: mass spectrometry peak pattern of Sj13Y19A.

Fig. 19. Detection results of activated partial thromboplastin time (APTT) of recombinant Sj13 mutant: X-axis: concentration of recombinant Sj13 mutant (μg/ml); Y-axis: activated partial thromboplastin time (sec). The final detection concentrations of Sj13R17A and Sj13Y19A: 0 μg/ml, 1.04 μg/ml, 2.08 μg/ml, 4.17 μg/ml, 8.33 μg/ml, 16.67 μg/ml, 33.33 μg/ml.

Figure 20. Test results of anticoagulant function of Sj13 protein and its mutants: A: Test results of endogenous coagulation pathway of Sj13, Sj13R17A and Sj13Y19A at different concentrations; B: When the final concentrations of Sj13, Sj13R17A and Sj13Y19A were all at 33.33 μg/ml, APTT test results. C and D are the detection results of different concentrations of Sj13, Sj13R17A and Sj13Y19A, FⅪa and FXa, respectively; E: the concentration of Sj13 and different mutants when the inhibition rate of FⅪa and FXa reaches 50% (IC ₅₀ ). X-axis in A, C, and D: different concentrations of Sj13, Sj13R17A, and Sj13Y19A; red, blue, and purple smooth lines represent the anticoagulant function test results of Sj13, Sj13R17A, and Sj13Y19A, respectively. Y-axis in A: activated partial thrombin time, Y-axis in C and D represent FⅪa and FXa residual enzyme percentages, respectively. X-axis of B: the final concentration of Sj13, Sj13R17A and Sj13Y19A; Y-axis: the prolongation time of activated partial thrombin when the final concentration of Sj13, Sj13R17A and Sj13Y19A is 33.33 μg/ml. X-axis of E: different final concentrations of blood coagulation factors, Y-axis: concentration of Sj13 and its mutants when the inhibition rate of blood coagulation factors reaches 50% (IC50).

Figure 21. Sj13Y19A prothrombin (PT) detection results: X-axis: Sj13Y19A concentration (μg/ml); Y-axis: activated partial thromboplastin time (sec). The different detection concentrations of Sj13Y19A were 0μg/ml, 1.04μg/ml, 2.08μg/ml, 4.17μg/ml, 8.33μg/ml, 16.67μg/ml, 33.33μg/ml.

Detailed ways

The present invention is further described in detail through the following examples, but it should be understood that the present invention is not limited by the following content.

Embodiment 1: Construction of Schistosoma japonicum polypeptide vector

1. Sj13PCR primer design

(1) Through the combination of structural biology and bioinformatics, a new Schistosoma japonicum gene/protein was determined from the Schistosoma japonicum gene/protein library, named Sj13, and its polypeptide amino acid sequence is as follows:

ETLKRYCNLPSDEGICRGYFRRYFYNVTSGECEVFYYGGCLGNRNRFSTIEKCWWYCKGL (SEQ ID NO. 1)

The protein sequence contains 6 cysteines, which can form 3 pairs of disulfide bonds, which are CysⅠ-CysVI, CysII-CysIV and CysIII-CysⅤ. This pairing mode is a typical feature of protein structure.

(2) Obtain the cDNA sequence of Sj13 through the sequence reverse translation website, as follows:

gaaaccctgaaacgctattgcaacctgccgagcgatgaaggcatttgccgcggctattttcgccgctatttttataacgtgaccagcggcgaatgcgaagtgttttattatggcggctgcctgggcaaccgcaaccgctttagcaccatgaaaaatgctggtggtattgcaaaggcctg (SEQ ID NO. 2)

(3) Use primer design software to design primers, as follows:

Sj13-FP1: CTATTTTCGCCGCTATTTTTATAACGTGACCAGCGGCGAATGCGAA (SEQ ID NO. 3)

Sj13-RP1: GGTTGCCCAGGCAGCCGCCATAATAAAACACTTCGCATTCGCCGCT (SEQ ID NO. 4)

Sj13-FP2: ACCTGCCGAGCGATGAAGGCATTTGCCGCGGCTATTTTCGCCGCTA (SEQ ID NO. 5)

Sj13-RP2: CAGCATTTTTCAATGGTGCTAAAGCGGTTGCGGTTGCCCAGGCAGC (SEQ ID NO. 6)

Sj13-FP3: CTGCATATGGAAACCCTGAAACGCTATTGCAACCTGCCGAGCGATG (SEQ ID NO. 7)

Sj13-RP3: GTGCTCGAGTCACAGGCCTTTGCAATACCACCAGCATTTTTCAATG (SEQ ID NO. 8)

The double underline indicates the nucleic acid sequence of the NdeI restriction site, and the single underline indicates the nucleic acid sequence of the XhoI restriction site.

2. Acquisition of Sj13DNA fragment

The 6 designed primers were amplified by 3 rounds of PCR using the overlap PCR method to amplify the complete gene sequence: in the first round of PCR, primers FP1 and RP1 were used for PCR amplification; The product and primers FP2 and RP2 were amplified; in the third round of PCR, the amplified product of the second round of PCR and primers FP3 and RP3 were used for amplification. The specific reaction system is shown in the table below:

PCR reaction conditions: repeat ②-④ 18 cycles in the first round, repeat ②-④ 28 cycles in the second and third rounds.

step temperature(°C) time ① Pre-denaturation 95 5 minutes ② denaturation 95 30 seconds ③ Annealing 55 30 seconds ④ extension 72 30 seconds ⑤ extend 72 10 minutes

3. Purification of Sj13 DNA fragment

After the PCR amplification product is purified, the size of the target DNA fragment is detected by agarose gel electrophoresis. The size of all target DNA fragments is about 200bp, which is basically consistent with the actual DNA fragment size of known Sj13.

4. Restriction endonuclease digestion of pET-28a(+) plasmid and purified Sj13DNA fragment

The purified Sj13DNA fragment and the pET-28a(+) plasmid were subjected to double digestion with restriction endonucleases NdeI and XhoI, respectively. The reaction systems are shown in the table below, and the reaction solution was placed in a constant temperature water bath at 37°C overnight (12-16 hours).

5. Purification and connection of digested pET-28a plasmid and Sj13DNA fragment

Add the pET-28a plasmid and Sj13 DNA fragment solution digested by the purified restriction endonuclease according to the following system, and put the mixed solution in a water bath at 22°C for 1 hour.

components Volume (μl) Digested and purified DNA fragments 6 Digested and purified pET-28a plasmid 2 T4 ligase 1 Buffer(10×) 1 total capacity 10

6. Transformation of pET-28a-Sj13 DNA fragment ligation products and identification of positive recombinants

Take 1 μl of cultured bacterial liquid as a template for PCR. Negative and positive controls were also set up. Negative control: 1 μl sterilized ultrapure water, positive control: 1 μl purified Sj13DNA fragment. Perform PCR detection according to the aforementioned reaction conditions. The size of the PCR product was detected by 2% agarose gel electrophoresis, and positive clones were selected and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for DNA sequencing. After the sequencing results are analyzed, the positive clone strains whose target nucleic acid sequence of the inserted fragment is completely consistent with the coding sequence of the known schistosome protein are expanded and cultured, the plasmid is extracted and the strain is preserved.

Embodiment 2: Expression and purification of Schistosoma japonicum polypeptide

The constructed pET-28a plasmid was transformed into E.coli Transetta (DE3) expression strains for strain expansion culture to obtain protein inclusion bodies. After the inclusion body was washed, it was denatured, diluted and refolded, concentrated by ultrafiltration and high-performance liquid chromatography (HPLC) to obtain a purified protein solution of the recombinant Schistosoma japonicum protein Sj13. The separation results of high performance liquid chromatography are shown in Fig. 2. High performance liquid chromatograph, obvious Sj13 single peak appears, and the protein liquid flowing out under its peak time is all collected, and the purified protein liquid collected is frozen into dry powder with a lyophilizer.

Embodiment 3: BCA quantification of Sj13 protein purification solution

The BCA protein concentration determination kit (Shanghai Biyuntian Biotechnology Co., Ltd.) was used to quantify the BCA of the purified Sj13 protein solution, and the detected protein concentration was calculated. 10 μg of protein powder was taken out and sent to the Institute of Chemistry, Chinese Academy of Sciences for mass spectrometry detection to further identify whether the protein was recombined successfully. The mass spectrometric identification results are shown in Figure 3. The mass spectrometry test results provided by the Institute of Chemistry, Chinese Academy of Sciences showed that the molecular weight of the recombinant Sj13 was 9375.3Da; the theoretical molecular weight of Sj13 predicted by the ExPASy-ProtParam tool (http://web.expasy.org/protparam/) website was 9380.52Da. The actual detected value of Sj13 mass spectrometry is consistent with the theoretical value.

Example 4: Detection of activated partial thrombin time (APTT)

Coagulation factor XII (FⅪ) in plasma can be activated by contact with anionic surfaces, and active FⅫ (FⅫa) in turn activates coagulation factor XI (FⅪ) to become active FⅪ (FⅪa), which in turn activates coagulation factor IX (FIX), active FIX (FIXa) activates coagulation factor X (FX) with the participation of phospholipids (PL), calcium ions (Ca2+) and active coagulation factor VIII (FⅧa), and the intrinsic coagulation pathway thus is activated. The APTT detection experiment is designed according to the activation principle of the endogenous blood coagulation pathway. The APTT detection reagent provides the ellagic acid, phospholipids and high concentration of calcium ions that are required to activate the endogenous blood coagulation system but not available in normal plasma. (Ca2+). Adding the APTT detection reagent to the plasma to be tested will immediately start the endogenous coagulation system, which will eventually lead to the formation of fibrin polymers and achieve the purpose of detection.

Sj13 was detected using a partial thromboplastin time (APTT) kit (Meide Pacific (Tianjin) Biotechnology Co., Ltd.). Each sample was tested 3 times. GraphPad Prism 7 software was used to analyze the data using one-way ANOVE, and a scatter diagram was drawn. P<0.05 is statistically significant.

Through APTT detection, it was found that the recombinant schistosome protein Sj13 could prolong the APTT detection time (Fig. 4), and at the concentration of 33.33 μg/ml, the prolongation time of Sj13 was 4.4 times that of the negative control. That is to say, Sj13 has an inhibitory effect on the endogenous blood coagulation system and has an anticoagulant function.

Embodiment 5: Detection of plasma prothrombin time (PT)

Coagulation factor VII (FVII) in plasma combines with tissue factor (III) released into the blood to form a complex after tissue injury, and is activated with the participation of PL and Ca2+, and then directly activates coagulation factor X (FX) to make it Become active FX (FXa), the extrinsic coagulation pathway is thus activated. The PT detection reagent provides the (PL) and high concentration of Ca2+ that are needed to activate the extrinsic blood coagulation system but not available in normal plasma. The exogenous coagulation system will start immediately when the PT detection reagent is added to the plasma to be tested, which will eventually lead to the formation of fibrin polymers and achieve the purpose of detection.

Sj13 was detected using a prothrombin time (PT) kit (Meide Pacific (Tianjin) Biotechnology Co., Ltd.). Each sample was tested 3 times. GraphPad Prism 7 software was used to analyze the data using one-way ANOVE, and a scatter diagram was drawn. P<0.05 is statistically significant.

The PT detection results of Sj13 protein are shown in FIG. 5 . The PT detection results showed that Sj13 failed to prolong the PT detection time, that is, the PT detection time was within the normal reference value range of 12-16 seconds. In other words, Sj13 has no inhibitory effect on the extrinsic coagulation system, and cannot prevent blood coagulation caused by the activation of the extrinsic coagulation system.

Embodiment 6: Detection of plasma thrombin time (TT)

Both the intrinsic and extrinsic coagulation pathways activate FX, thereby converting prothrombin (II) into thrombin (IIa), and thrombin can convert fibrinogen into soluble fibrin monomers. With the participation of (FXIII) and high concentration of Ca ²⁺ , soluble fibrin monomers form firm insoluble fibrin polymers, which is the common way of blood coagulation. The TT detection reagent directly provides thrombin to convert the fibrinogen in the plasma into fibrin directly, and detects the time it takes.

Sj13 was detected using a thrombin time (TT) kit (Meide Pacific (Tianjin) Biotechnology Co., Ltd.). Each sample was tested 3 times. GraphPad Prism 7 software was used to analyze the data using one-way ANOVE, and a scatter diagram was drawn. P<0.05 is statistically significant.

The TT detection results of Sj13 protein are shown in FIG. 6 . The test results showed that the TT detection time was within the normal reference range of 11-18 seconds, proving that Sj13 does not affect the common pathway of the blood coagulation system and cannot prevent blood coagulation caused by thrombin activation of fibrinogen.

Example 7: Verification of Antithrombotic Function of Schistosoma japonicum Anticoagulant Polypeptide Sj13

1. Construction of FeCl3-injured common carotid artery thrombus model in C57BL/6 mice

24 normally raised C57BL/6 mice were randomly divided into low-concentration inhibitor group (n=6), high-concentration inhibitor group (n=6), positive control group (n=6), negative control group (n=6) =6). The concentration of the inhibitor in the low concentration group is 0.5mg/kg, the concentration of the inhibitor in the high concentration group is 1mg/kg, the administration concentration of heparin sodium in the positive control group is 3.6mg/kg, and the concentration of normal saline in the negative control group is 0.154M .

After C57BL/6 mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (4ml/kg), they were fixed supine on a fixture. Use a warm air blower to blow the rat tail, and after the blood vessels are filled, tie the proximal end of the rat tail tightly with a rubber band. Inject 0.1ml of different concentrations of inhibitors, unfractionated heparin and normal saline through the tail vein. After 15 minutes, the neck was disinfected with 75% alcohol, and the skin was cut in the middle of the neck, the soft tissue and muscle were separated layer by layer, and the right common carotid artery was exposed, and 2×1mm filter paper was soaked in 10% FeCl3 solution and wrapped in the common carotid artery. arterial surface, removed after 3 minutes. After 15 minutes of observation, the mice were killed immediately, and the thrombus-forming common carotid artery was taken out, rinsed with normal saline, blotted dry with filter paper, weighed and measured the length of the thrombus, and fixed with 4% paraformaldehyde solution.

2. Construction of C57BL/6 mouse tail docking model

Eighteen normally fed C57BL/6 mice were randomly divided into inhibitor group (n=6), positive control group (n=6) and negative control group (n=6). The concentration of the inhibitor was 10 mg/kg, the administration concentration of unfractionated heparin in the positive control group was 100 mg/kg, and the concentration of normal saline in the negative control group was 0.154M.

After C57BL/6 mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (4ml/kg), they were fixed supine on a fixture. Use a warm air blower to blow the rat tail, and after the blood vessels are filled, tie the proximal end of the rat tail tightly with a rubber band. Inject 0.1ml of different concentrations of inhibitors, unfractionated heparin and normal saline through the tail vein. After 15 minutes, cut off 3mm from the top of the mouse tail and immediately count the time to observe the bleeding. The observation of the experimental results shall not exceed 30 minutes.

3. Anticoagulant function evaluation of FeCl3-injured common carotid artery thrombus model in Sj13C57BL/6 mice

The mouse FeCl3 thrombus model is an animal thrombus model based on the chemical injury method. External application of FeCl3 solution to blood vessel walls causes high-priced iron ions to infiltrate into blood vessels to cause oxidation reactions, damage vascular endothelial cells, expose subendothelial collagen, promote platelet adhesion, release and aggregation reactions, activate the coagulation system, and form thrombus. The mouse FeCl3 thrombus model is widely used in the screening and pharmacodynamic evaluation of hemorrhagic diseases, the pathogenesis of thrombosis and thromboembolic diseases, pathophysiological changes, and anticoagulant drugs.

In the present invention, 100 μl of 0.154M NaCl (normal saline), sodium heparin (3.6 mg/kg), low concentration Sj13 (0.5 mg/kg), and high concentration Sj13 (1.0 mg/kg) are respectively injected through the tail vein. 15 minutes after the injection, 10% FeCl3 solution was applied externally to the right common carotid artery of the mouse for 3 minutes. After 15 minutes of observation, the isolated and exposed common carotid artery was taken out, its length was measured, and the wet weight of the thrombus was weighed. It was fixed with 4% paraformaldehyde . The results of data analysis are shown in Fig. 7; the macroscopic pictures of Sj13 affecting the thrombosis of FeCl3-injured common carotid artery in mice are shown in Fig. 8a-8d.

Sj13 low-concentration group (0.5mg/kg), high-concentration group (1.0mg/kg) and heparin sodium (3.6mg/kg) group had a very significant inhibitory effect on thrombus formation compared with the data of the 0.154M NaCl group (P<0.0001 ); Sj13 low-concentration group, high-concentration group and heparin sodium group were compared with each other, and there was no obvious difference in the effect of inhibiting thrombus formation (P>0.05), as shown in Figure 7.

In the group injected with 0.154M NaCl, the isolated common carotid artery was observed with the naked eye, and it was found that the lumen of the common carotid artery was blocked by the formed thrombus, showing a dark red color. This phenomenon proves that the model of common carotid artery thrombosis induced by FeCl3 in mice is successful. It can also be observed from the picture shown in Figure 8a, which lays the foundation for further evaluation of the anticoagulant function of Sj13 in vivo.

As a traditional anticoagulant drug, heparin sodium is widely used in clinical thrombosis prevention and treatment, and is suitable as a positive control drug to participate in the evaluation of anticoagulant function in animal models of thrombosis. In the heparin sodium injection group, the weight of the isolated common carotid artery was significantly lower than that in the 0.154M NaCl injection group; it was observed with the naked eye that the damaged common carotid artery lumen was partially collapsed due to lack of thrombus support; There are a small amount of light-transmitting regions, see Figure 8b. It has been confirmed that heparin sodium has an effect on the thrombosis of common carotid artery injured by FeCl3 in mice, and can significantly inhibit the formation of thrombus. On the other hand, the modeling stability of the thrombus animal model was once again verified.

In the process of weighing and observing the isolated common carotid arteries in the low concentration group and high concentration group of Sj13, the situation similar to that of the positive control drug-heparin sodium appeared: the wet weight of the thrombus was lighter, and the common carotid artery blood vessels were partially collapsed and The existence of partially light-transmitting regions is more obvious in the high-concentration group, as shown in Figures 8c-8d. Sj13 has the effect of inhibiting the thrombosis of common carotid artery injured by FeCl3 in mice, and the effect is similar to the effect of injecting 3.6mg/kg heparin sodium.

4. Evaluation of bleeding risk in Sj13C57BL/6 mouse tail docking model

The in vivo anticoagulant function of Sj13 was evaluated by the common carotid artery thrombosis model injured by FeCl3 in mice, and it was found that it also has anticoagulant function in vivo and can inhibit thrombus formation. In other words, Sj13, like other anticoagulant drugs, faces the risk of massive bleeding during anticoagulation. The mouse tail docking model is also widely used to evaluate the pharmacodynamics of various anticoagulant drugs and observe the side effects of bleeding.

In the present invention, 100 μl of 0.154M NaCl, heparin sodium (100 mg/kg) and Sj13 (10 mg/kg) are respectively injected through the tail vein. After 15 minutes of injection, cut the time immediately after cutting 3mm from the top of the mouse tail, and observe the bleeding situation. Sj13 group (10mg/kg) and 0.154M NaCl group were significantly less bleeding risk than heparin sodium (100mg/kg) group (P<0.0001); Sj13 group and 0.154M NaCl group were compared with each other, there was no bleeding risk Significant difference (P>0.05). The analysis of the results is shown in Figure 9, and it can be preliminarily concluded that Sj13 has relatively low bleeding side effects.

Example 8: Research on the anticoagulant mechanism of Schistosoma japonicum anticoagulant polypeptide Sj13

1. Identification of coagulation factor inhibitory activity of rSj13:

The inhibitory activities of rSj13 were tested for fibrinolytic enzyme (Plasmin), thrombin (Thrombin), coagulation factor Ⅻa, coagulation factor Ⅺa, and coagulation factor FXa. Different concentrations of rSj13 have no inhibitory effect on FⅫa and thrombin, but have obvious concentration-dependent inhibitory effects on FⅪa, FXa and plasmin, with IC50 values of 63nM, 3.327nM, and 78.125nM, respectively. 1.25 μM rSj13 can inhibit FⅪa and plasmin by more than 90%; 19.53 nM Sj13 can also inhibit FXa by more than 90%. The results are shown in FIG. 10 .

2. In vitro enzyme kinetics identification of Sj13:

(1) Kinetic detection results of Sj13 on FⅪa enzyme

The double reciprocal plot of 1/V versus 1/[S] shows that the inhibitory effect of Sj13 on FⅪa is mixed inhibition, as shown in Figure 11A. Sj13 can not only bind to sites other than the active center of FⅪa, but also bind to the complex formed by FⅪa and FⅧa, thereby inhibiting the activity of FⅪa. The double reciprocal plot of Sj13 at 0nM concentration obtained the intercept and slope of the Y axis, and calculated the Km value of FⅪa as 0.66×10 ^-3 mol/L.

Whether Sj13 binds to FⅪa first or binds to the complex formed by FⅪa and FⅧa depends on the values of the inhibition constants Ki and Ki' of Sj13. The slopes of Sj13 at different concentrations and the intercepts on the Y-axis were plotted against the different concentrations of Sj13 to obtain Figures 11B and C. The values of Sj13Ki and Ki' were calculated using the double reciprocal equation derived from the Mie equation in the presence of competitive and anticompetitive inhibitors. If Ki<Ki', Sj13 binds before FⅪa, otherwise, it binds before the complex formed by FⅪa and FⅧa. The Ki value of Sj13 is equal to 423.91×10 ^-9 mol/L, and the Ki' value is equal to 55.49×10 ^-9 mol/L; Ki>Ki', Sj13 inhibits the activity of FⅪa by binding to the complex formed by FⅪa and FⅧa, blocking Activation of coagulation pathways.

(2) Sj13FXa enzyme kinetics detection results

By plotting 1/V against 1/[S] double reciprocal, the inhibitory effect of Sj13 on FXa is an anti-competitive inhibition, as shown in Figure 12A. Sj13 and FXa bind to the complex formed by FVa, thereby inhibiting the activity of FXa. Sj13 was plotted with double reciprocal at 0nM concentration to obtain the intercept and slope of the Y axis, and the calculated Km value of FXa was 4.47×10 ^-3 mol/L.

The combination of Sj13 and the complex formed by FXa and FVa depends on the value of the inhibition constant Ki of Sj13. The intercepts on the Y axis of Sj13 at different concentrations were plotted against the different concentrations of Sj13 to obtain Figure 12B. Sj13Ki values were calculated using the double reciprocal equation derived from the Mie equation in the presence of anti-competitive inhibitors. The Sj13Ki value is equal to 2.66×10 ^-9 mol/L, Sj13 inhibits the activity of FⅪa by binding to the complex formed by FXa and FVa, and blocks the activation of coagulation pathway.

3. Effect of digestive enzymes on Sj13

The effect of polypeptide Sj13 on two common intestinal digestive enzymes, trypsin and α-chymotrypsin, was preliminarily studied. The results showed that the polypeptide Sj13 had a good inhibitory effect on both trypsin and α-chymotrypsin, and the inhibition rate reached 50% at 233×10 ^-3 M and 48×10 ^-3 M respectively ( FIG. 13 ).

In summary, polypeptide Sj13 is a new anticoagulant with high stability and a unique anticoagulant mechanism. Its inhibition on FⅪa and FXa belongs to mixed inhibition and anti-competitive inhibition respectively, both of which are related to coagulation factors other than the active center. site binding to affect its coagulation function.

Example 9: Structure-activity relationship and mutant function analysis of anticoagulant polypeptide Sj13 of Schistosoma japonicum

1. Mutant design of Sj13

Polypeptide Sj13 consists of 57 amino acid residues, containing 6 cysteines (Cys), forming 3 pairs of disulfide bonds, namely Cys7–Cys57 (CI–CVI), Cys16–Cys40 (CII–CIV), Cys32– Cys53 (CⅢ–CⅤ), corresponding to the polypeptide BPTI; the whole molecule is pear-shaped, consisting of two antiparallel β-sheets, two α-helices, β-turns and some loops, similar to the structure of BPTI, as shown in Figure 14 .

Through the modeling of the three-dimensional structure of Sj13 protein, it was found that the amino acid residues at the P1 and P2′ sites stretched outwards, which were positively charged and benzene rings respectively, and were easy to react with other molecules. Accordingly, we selected the potential key active site P1 (R17) of the polypeptide Sj13, and the general active site P2' (Y19), and selected the positively charged basic amino acid on the P1 site according to the characteristics of the inhibitor active site. Arginine (Arg) is mutated to nonpolar aliphatic alanine (Ala), and the nonpolar aromatic amino acid tyrosine (Try) at the P2′ site is mutated to nonpolar aliphatic alanine (Ala). The mutation site of the amino acid sequence of Sj13 is shown in FIG. 15 . The three-dimensional structures of Sj13 protein and its mutants are shown in FIG. 16 . Alanine mutant studies were performed by re-testing the function of the Sj13 mutants Sj13R17A and Sj13Y19A.

Sj13R17A: ETLKRYCNLPSDEGICAGYFRRYFYNVTSGECEVFYYGGCLGNRNRFSTIEKCWWYCKGL (SEQ ID NO. 9)

Sj13Y19A:ETLKRYCNLPSDEGICRGAFRRYFYNVTSGECEVFYYGGCLGNRNRFSTIEKCWWYCKGL (SEQ ID NO. 10)

2. Expression, purification and identification of Sj13 mutant

High-performance liquid chromatography, after running for about 10-15 minutes, the recombined Sj13R17A and Sj13Y1719A showed obvious single peaks at 21.94 minutes and 20.786 minutes respectively, and all the protein liquids flowing out at the peak time were collected and freeze-dried The machine freezes the collected purified protein into dry powder, quantifies the protein with the BCA kit, and stores it in a -80°C refrigerator. 10 μg of protein powder was taken out and sent to the Institute of Chemistry, Chinese Academy of Sciences for mass spectrometry detection to further identify whether the protein was recombined successfully. The results of mass spectrometry identification are shown in Figure 17.

The mass spectrometry results provided by the Institute of Chemistry, Chinese Academy of Sciences (Figure 18) show that the molecular weights of the recombinant Sj13R17A and Sj13Y1719A are 9287.1Da and 9281.4Da respectively; The predicted theoretical molecular weights of Sj13R17A and Sj13Y1719A are 9295.41Da and 9288.42Da, respectively. The difference between the actual detection value and the theoretical value is very small, and the recombinant Sj13 mutant is successfully recombined, but what changes will occur in its function remains to be further tested.

3. Detection of endogenous coagulation function of Sj13 mutant

Both Sj13R17A and Sj13Y19A can prolong the detection time of APTT (Figure 19), and when the concentration is 33.33 μg/ml, the prolongation time is 2.3 and 17.8 times that of the negative control, respectively. That is to say, the recombinant Sj13 mutation still has an inhibitory effect on the endogenous blood coagulation system.

4. Identification of coagulation factor inhibitory activity of Sj13 mutant

The inhibitory effect of Sj13 mutant on FXa was obviously concentration-dependent, the IC50 values of Sj13R17A and Sj13Y19A were 189nM and 6.022nM respectively; the inhibitory effect on FⅪa was also obvious concentration-dependent, the IC50 values of Sj13R17A and Sj13Y19A were 792nM and 20nM ( Figure 20A,B). However, the inhibitory effects of Sj13Y19A on FXa and FⅪa were superior to those of Sj13R17A (when the inhibition rate reached 50%), which were 31.38 and 39.6 times respectively (Fig. 20C).

It was found by APTT that the two mutants of Sj13 could inhibit the endogenous blood coagulation pathway. Compared with the highest inhibitory concentration of Sj13, Sj13Y19A>Sj13>Sj13R17A, but the activity of Sj13R17A has been weakened by 1 times, and the activity of Sj13Y19A has been enhanced by 3 times. (FIG. 20AB). The detection of FⅪa found that its IC50 value is Sj13Y19A<Sj13<Sj13R17A (Figure 20C); the IC50 value of FXa is Sj13<Sj13Y19A<Sj13R17A (Figure 20D). Regardless of FⅪa or FXa, the inhibitory activity of Sj13R17A was the lowest among the three, and the inhibitory activity of Sj13Y17A on FXa was only 1-fold lower than that of Sj13 (Fig. 20E).

5. Detection of exogenous coagulation function of Sj13Y19A

Sj13Y19A failed to delay the prothrombin time ( FIG. 21 ) and did not affect the extrinsic coagulation pathway.

In summary, the coagulation factor P1 site is biased towards positively charged basic amino acids, mainly arginine (Arg), including FⅪa and FXa; the P2′ site is biased towards non-polar amino acids. If the sites of coagulation factors P1 and P2' are changed, their activation will be affected [56]. The characteristics of the amino acid residues at the P1 and P2′ sites of Sj13 are similar to those of coagulation factors. After the arginine (Arg) corresponding to the P1 site is mutated into the non-polar aliphatic amino acid alanine (Ala), the anticoagulant activity of Sj13R17A It was indeed weakened, that is to say, the change of the properties of amino acid residues caused the reduction of the ability of Sj13R17A to fit FⅪa and FXa. In contrast, mutating the tyrosine (Tyr) residue at the Sj13P2' site to a tyrosine (Ala), the anticoagulant activity of Sj13Y19A without the phenyl ring was enhanced. The effect of inhibiting FⅪa was slightly different from that of wild-type Sj13, and the effect of inhibiting FXa was slightly better (Fig. 20E), which greatly enhanced the effect of inhibiting the endogenous coagulation pathway.

It can be preliminarily inferred that the arginine (Arg) on the P1 site plays an important role in Sj13 inhibiting the endogenous blood coagulation process. The non-polar amino acid residues at the P2′ site have hydrophilic or hydrophobic characteristics, which will have a certain impact on its activity, which lays the foundation for in-depth elucidation of the structure-activity relationship of the polypeptide Sj13 and the molecular design based on the polypeptide Sj13.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

SEQUENCE LISTING

<110> Hubei University of Medicine

<120> Sj13 polypeptide and its application in the preparation of antithrombotic drugs

<130> CP11802088C

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 60

<212> PRT

<213> Sj13

<400> 1

Glu Thr Leu Lys Arg Tyr Cys Asn Leu Pro Ser Asp Glu Gly Ile Cys

1 5 10 15

Arg Gly Tyr Phe Arg Arg Tyr Phe Tyr Asn Val Thr Ser Gly Glu Cys

20 25 30

Glu Val Phe Tyr Tyr Gly Gly Cys Leu Gly Asn Arg Asn Arg Phe Ser

35 40 45

Thr Ile Glu Lys Cys Trp Trp Tyr Cys Lys Gly Leu

50 55 60

<210> 2

<211> 180

<212>DNA

<213> cDNA of Sj13

<400> 2

gaaaccctga aacgctattg caacctgccg agcgatgaag gcatttgccg cggctatttt 60

cgccgctatt tttataacgt gaccagcggc gaatgcgaag tgttttatta tggcggctgc 120

ctgggcaacc gcaaccgctt tagcaccatt gaaaaatgct ggtggtattg caaaggcctg 180

<210> 3

<211> 46

<212>DNA

<213> Sj13-FP1

<400> 3

ctatttttcgc cgctattttt ataacgtgac cagcggcgaa tgcgaa 46

<210> 4

<211> 46

<212>DNA

<213> Sj13-RP1

<400> 4

ggttgcccag gcagccgcca taataaaaca cttcgcattc gccgct 46

<210> 5

<211> 46

<212>DNA

<213> Sj13-FP2

<400> 5

acctgccgag cgatgaaggc atttgccgcg gctattttcg ccgcta 46

<210> 6

<211> 46

<212>DNA

<213> Sj13-RP2

<400> 6

cagcattttt caatggtgct aaagcggttg cggttgccca ggcagc 46

<210> 7

<211> 46

<212>DNA

<213> Sj13-FP3

<400> 7

ctgcatatgg aaaccctgaa acgctattgc aacctgccga gcgatg 46

<210> 8

<211> 46

<212>DNA

<213> Sj13-RP3

<400> 8

gtgctcgagt cacaggcctt tgcaatacca ccagcatttt tcaatg 46

<210> 9

<211> 60

<212> PRT

<213> Sj13R17A

<400> 9

Glu Thr Leu Lys Arg Tyr Cys Asn Leu Pro Ser Asp Glu Gly Ile Cys

1 5 10 15

Ala Gly Tyr Phe Arg Arg Tyr Phe Tyr Asn Val Thr Ser Gly Glu Cys

20 25 30

Glu Val Phe Tyr Tyr Gly Gly Cys Leu Gly Asn Arg Asn Arg Phe Ser

35 40 45

Thr Ile Glu Lys Cys Trp Trp Tyr Cys Lys Gly Leu

50 55 60

<210> 10

<211> 60

<212> PRT

<213> Sj13Y19A

<400> 10

Glu Thr Leu Lys Arg Tyr Cys Asn Leu Pro Ser Asp Glu Gly Ile Cys

1 5 10 15

Arg Gly Ala Phe Arg Arg Tyr Phe Tyr Asn Val Thr Ser Gly Glu Cys

20 25 30

Glu Val Phe Tyr Tyr Gly Gly Cys Leu Gly Asn Arg Asn Arg Phe Ser

35 40 45

Thr Ile Glu Lys Cys Trp Trp Tyr Cys Lys Gly Leu

50 55 60

Claims (10)

1. A polypeptide Sj13, characterized in that the polypeptide comprises the amino acid sequence shown in SEO ID NO.1. 2. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the polypeptide Sj13 according to claim 1; preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEO ID NO.2. 3. A polypeptide Sj13 mutant, characterized in that the mutant has an amino acid substitution or substitution at the P1 and/or P2' site of the Sj13 polypeptide, and the P1 site corresponds to R17 of SEQ ID NO.1 , the P2' site corresponds to Y19 of SEQ ID NO.1; preferably, the replacement or substitution of the P1 site is a basic amino acid residue; preferably, the replacement or substitution of the P2' site is a non-polar The characteristic hydrophobic amino acid residue; preferably, the polypeptide Sj13 mutant comprises the amino acid sequence shown in SEO ID NO.9 or 10. 4. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the polypeptide Sj13 mutant according to claim 3. 5. A vector, characterized in that the vector comprises the nucleic acid molecule according to claim 2 or 4. 6. A cell, characterized in that the cell comprises the polypeptide Sj13 of claim 1, the polypeptide Sj13 mutant of claim 3, the nucleic acid molecule of claim 2, and/or the nucleic acid molecule of claim 4 . 7. A set of primers, characterized in that the set of primers comprises 3 pairs of primers, respectively: Sj13-FP1 shown in SEO ID NO.3 and Sj13-RP1 shown in SEO ID NO.4; Sj13-FP2 shown in SEO ID NO.5 and Sj13-RP2 shown in SEO ID NO.6; Sj13-FP3 shown in SEO ID NO.7 and Sj13-RP3 shown in SEO ID NO.8. 8. The use of the primer according to claim 7 in amplifying the nucleic acid molecule encoding the polypeptide Sj13. 9. Use of the polypeptide according to claim 1, the mutant according to claim 3, and/or the nucleic acid molecule according to claim 2 or 4 in the preparation of anticoagulant and antithrombotic preparations; preferably, the preparation is A kit or a pharmaceutical composition, more preferably a pharmaceutical composition. 10. The use of the polypeptide Sj13 of claim 1 and/or the mutant of the polypeptide Sj13 of claim 3 in inhibiting coagulation-related factors XIa, Xa and/or Plasmin.