Polypeptide from Buthus martensii Karsch and application thereof

Abstract

The invention discloses a scorpion-derived polypeptide and application thereof, and belongs to the technical field of functional proteins. The amino acid sequence of the polypeptide derived from the Buthus martensii Karsch is as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5. The polypeptide derived from the Buthus martensii Karsch is a polypeptide derived from the Buthus martensii Karsch. The invention also provides an application of the buthus martensii-sourced polypeptide with the amino acid sequence shown as SEQ ID NO. 1 in preparation of a blood coagulation promoting drug and an application of the buthus martensii-sourced polypeptide with the amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 in preparation of a blood coagulation promoting drug. The buthus martensii-sourced polypeptide provided by the invention has a relatively good application value in the aspect of research and development of antithrombotic drugs or coagulant drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of functional proteins, and particularly to a polypeptide derived from Buthus martensii Karsch and its application. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The coagulation system activates prothrombin through two pathways, the intrinsic and extrinsic pathways, promoting the conversion of fibrinogen to fibrin, while the anticoagulation system relies on antithrombin-III, the protein C pathway, and the fibrinolytic system for dynamic regulation. The dynamic balance between blood coagulation and the anticoagulation system is the key to maintaining normal vascular function: when the balance is disrupted, excessive enhancement of coagulation function can trigger thrombosis and cardiovascular events, while insufficient coagulation function may lead to severe bleeding, such as deep bleeding during surgery, both of which seriously threaten health. Therefore, the development of safe and effective anticoagulant and procoagulant drugs is of great significance for maintaining coagulation balance and ensuring clinical treatment.

[0004] Existing anticoagulant drugs have significant limitations: conventional chemical drugs such as warfarin require strict restriction of vitamin K intake or rely on hepatic enzyme metabolism, and are prone to interact with other drugs; although polypeptide drugs have high specificity and simple metabolism, some have side effects such as thrombocytopenia and bleeding, while natural-source anticoagulant peptides are difficult to produce on a large scale due to low content and high purification costs. In terms of procoagulant drugs, there is an urgent clinical need. Although commonly used snake venom thrombin (such as reptilase) has the advantages of rapid onset and significant efficacy, it relies on specific snake species resources and may face resource limitations or immunogenicity risks in long-term use, and there is an urgent need to develop new procoagulant drugs.

[0005] As a traditional Chinese medicine, whole scorpion has the effects of calming endogenous wind and stopping convulsions, dredging collaterals and relieving pain, and is widely used in the treatment of diseases such as infantile convulsions and hemiplegia. Its main medicinal variety is Buthus martensii Karsch (also known as Mesobuthus martensii). At present, active ingredients with regulatory effects on blood coagulation function have been found in the venom of Buthus martensii Karsch. However, existing technologies mostly focus on crude extracts or mixed components of Buthus martensii Karsch, and there are few reports on single polypeptides with clear sequences and significant activities, which are difficult to meet the clinical needs for the diversified development of anticoagulant and procoagulant drugs. Summary of the Invention

[0006] In view of this, the present invention provides a polypeptide derived from Buthus martensii Karsch and its application. Five polypeptides, MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126, were identified from the gene transcriptome of Buthus martensii Karsch, and the recombinant polypeptides obtained by prokaryotic expression of their sequences showed procoagulant or anticoagulant activities in in vitro tests.

[0007] In a first aspect, the present invention provides a polypeptide derived from Buthus martensii Karsch, and the amino acid sequence of the polypeptide derived from Buthus martensii Karsch is as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5.

[0008] Specifically, the amino acid sequence of MmTX70 (SEQ ID NO. 1) is as follows: QLVTGCVFWQCRNSCLSKGYRGGGMCQGIIEKKCYCIR.

[0009] The amino acid sequence of MmTX118 (SEQ ID NO. 2) is as follows: KKNGYPADDDGCKIACFFRKNGCPFACQKLNGSTGSCDIVKKACKCEGLPDNAKLWDQTKQCNKK.

[0010] The amino acid sequence of MmTX67 (SEQ ID NO. 3) is as follows: KTSCNHLKRCAKYGFYRNCTECCKQHKHSGGYCTMYKCLCKI.

[0011] The amino acid sequence of MmTX106 (SEQ ID NO. 4) is as follows: GDGYIRNVDDGCKLSCFLQNEMCNRECKIRGAYYGYCWSWGISCWCEGLPDNKLWKQETNTCRGKK.

[0012] The amino acid sequence of MmTX126 (SEQ ID NO. 5) is as follows: SMFDDGYPVKNGCRISCIPDEHDDLCEQFCKKNKAETGGCDFDADACKCWGELGGMEIWEPKSSECKSWNDNLITKILEN.

[0013] In a second aspect, the present invention provides a coding gene that encodes the above-mentioned polypeptide derived from Buthus martensii Karsch, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

[0014] Specifically, the nucleotide sequence encoding MmTX70 (SEQ ID NO. 6) from the 5'-end to the 3'-end is: CAGCTTGTGACCGGTTGTGTTTTCTGGCAGTGTCGTAATAGCTGTCTGAGCAAAGGCTATCGCGGTGGTGGTATGTGCCAGGGTATTATTGAAAAGAAATGCTACTGCATCCGC。

[0015] The nucleotide sequence encoding MmTX118 (SEQ ID NO. 7) from the 5'-end to the 3'-end is: AAAAAGAATGGTTATCCAGCAGATGATGATGGTTGTAAGATAGCTTGCTTTTTCCGTAAGAATGGTTGCCCTTTTGCATGCCAAAAACTGAATGGCAGTACTGGTTCTTGTGATATAGTGAAAAAAGCATGCAAATGTGAAGGACTTCCAGATAATGCAAAACTTTGGGATCAAACCAAACAATGCAACAAAAAATAA。

[0016] The nucleotide sequence encoding MmTX67 (SEQ ID NO. 8) from the 5'-end to the 3'-end is: AAAACTAGCTGCAATCATTTAAAGCGATGTGCAAAGTATGGATTTTATCGAAATTGTACCGAATGCTGTAAACAACATAAACATTCAGGAGGTTATTGTACAATGTATAAATGCCTATGTAAAATATAA。

[0017] The nucleotide sequence encoding MmTX106 (SEQ ID NO. 9) from the 5'-end to the 3'-end is: GGTGACGGTTATATTCGTAATGTTGATGATGGCTGCAAGCTGAGCTGTTTTCTGCAGAATGAAATGTGCAATCGTGAATGTAAGATCCGTGGTGCCTATTATGGCTATTGCTGGAGCTGGGGTATTAGTTGCTGGTGCGAAGGCCTGCCGGATAATAAGCTGTGGAAACAGGAAACCAATACCTGTCGCGGTAAAAAATAA。

[0018] The nucleotide sequence encoding MmTX126 (SEQ ID NO. 10) from the 5'-end to the 3'-end is: TCAATGTTCGATGATGGATATCCAGTTAAAAACGGCTGCCGAATTTCATGCATACCAGATGAGCATGATGATCTTTGCGAACAATTCTGCAAGAAGAATAAAGCAGAAACAGGTGGATGTGACTTTGATGCAGATGCATGCAAATGTTGGGGAGAATTAGGTGGAATGGAAATATGGGAACCTAAATCATCAGAATGCAAGAGTTGGAATGACAATCTAATAACAAAGATTTTGGAAAATTAA。

[0019] The present invention has no special restrictions on the source of the encoding gene, and it can be prepared by conventional methods in the art or synthesized by biological companies.

[0020] In the third aspect, the present invention provides a recombinant plasmid into which the above-mentioned encoding gene is inserted.

[0021] In the present invention, the original plasmid of the recombinant plasmid includes pET-32a.

[0022] In the present invention, the recombinant plasmid is preferably constructed by the following method: ligating the encoding gene into the vector pET-32a; the method of ligating the encoding gene into the vector pET-32a is preferably homologous recombination to construct the plasmid.

[0023] In the fourth aspect, the present invention provides a recombinant bacterium containing the above-mentioned recombinant plasmid.

[0024] In the present invention, the original bacterium of the recombinant bacterium is preferably Escherichia coli, more preferably Escherichia coli BL21(DE3) or Escherichia coli Shuffle T7 strain.

[0025] In the fifth aspect, the present invention provides a procoagulant pharmaceutical composition comprising a polypeptide derived from Buthus martensii Karsch with the amino acid sequence of SEQ ID NO. 1.

[0026] In the sixth aspect, the present invention provides the use of the above-mentioned procoagulant pharmaceutical composition in the preparation of a procoagulant drug.

[0027] In the seventh aspect, the present invention provides an anticoagulant pharmaceutical composition comprising a polypeptide derived from Buthus martensii Karsch with the amino acid sequence shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5.

[0028] In the eighth aspect, the present invention provides the use of the above-mentioned anticoagulant pharmaceutical composition in the preparation of an anticoagulant drug.

[0029] In a ninth aspect, the present invention provides the use of a polypeptide derived from Buthus martensii Karsch with an amino acid sequence as shown in SEQ ID NO. 1 in the preparation of a procoagulant drug.

[0030] In a tenth aspect, the present invention provides the use of a polypeptide derived from Buthus martensii Karsch with an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 in the preparation of an anticoagulant drug.

[0031] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention has for the first time identified and successfully constructed a prokaryotic expression system for five polypeptides, namely MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126, from the venom of Buthus martensii Karsch, breaking through the industrialization bottleneck of low content and difficult purification in the traditional natural extraction method, and being easy for large-scale production. The recombinant polypeptides obtained by heterologous expression in the present invention showed procoagulant activity or anticoagulant activity in experiments: among them, MmTX70 shortened the time for fibrin formation through the endogenous pathway and had a procoagulant effect; while the four polypeptides MmTX118, MmTX67, MmTX106, and MmTX126 had the activity of prolonging the fibrin formation time and the blood clot formation time, showing good anticoagulant effects, especially MmTX67 and MmTX106. The polypeptides derived from Buthus martensii Karsch provided by the present invention have good application value in the research and development of antithrombotic drugs or procoagulant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0033] Figure 1 It is a Tricine-SDS-PAGE result diagram of five polypeptides, namely MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126, in Example 1 of the present invention. Among them, A is the Tricine-SDS-PAGE result diagram of MmTX70, MmTX118, MmTX67, and MmTX126, and B is the Tricine-SDS-PAGE result diagram of MmTX106; Figure 2It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 2 of the present invention on activated partial thromboplastin time (APTT); Figure 3 It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 2 of the present invention on prothrombin time (PT); Figure 4 It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 2 of the present invention on thrombin time (TT); Figure 5 It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 3 of the present invention on recalcification time (25 μM); Figure 6 It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 3 of the present invention on recalcification time (50 μM); Figure 7 It is the determination result of the inhibitory effects of five scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Buthus martensii Karsch in Example 4 of the present invention on thrombosis; Detailed implementation manners

[0034] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0035] The technical solution of the present invention will be further elaborated below in conjunction with specific embodiments. The present invention has no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products well-known to those skilled in the art can be used.

[0036] In the following embodiments, Tricine-SDS-PAGE is a high-resolution electrophoresis technique for separating small molecule proteins or polypeptides, and its name is composed of the abbreviations of three core components (Tricine, SDS, PAGE). The meanings and functions of each part are as follows: Tricine represents N-tris(hydroxymethyl)methylglycine, which is an amphoteric ion buffer; SDS represents sodium dodecyl sulfate, which is an anionic surfactant; PAGE represents polyacrylamide gel electrophoresis.

[0037] Example 1 This example provides five polypeptides of Buthus martensii Karsch, namely MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126. These polypeptides are obtained through molecular cloning technology and prokaryotic expression and purification in Escherichia coli.

[0038] 1. Construction of recombinant plasmids Using the cDNA of Buthus martensii Karsch as a template, the nucleotide sequences of MmTX118, MmTX67, and MmTX126 are obtained by PCR (shown as SEQ ID NO. 7, 8, and 10 respectively), and are ligated to the pET32a vector through homologous recombination. The gene sequences of MmTX70 and MmTX106 are codon-optimized, and the optimized gene sequences are synthesized by gene synthesis technology (shown as SEQ ID NO. 6 and 9), and are respectively ligated to the pET32a vector to form plasmids. A recombinant enterokinase site (GACGACGACGACAAG, SEQ ID NO. 11) is introduced at the 5' end of each gene. The recombinant plasmids pET32a-MmTX70, pET32a-MmTX118, pET32a-MmTX67, pET32a-MmTX106, and pET32a-MmTX126 are constructed.

[0039] 2. Prokaryotic expression The above recombinant plasmids pET-32a-MmTX118, pET-32a-MmTX67, and pET32a-MmTX126 are transferred into Escherichia coli BL21(DE3) for expression; the recombinant plasmids pET-32a-MmTX70 and pET-32a-MmTX106 are transferred into Escherichia coli ShuffleT7 strain for expression. Single colonies are picked into shaking tubes, added with 5 mL of LB medium and 5 μL of 100 mg / mL ampicillin (AMP), and cultured overnight at 37 °C and 200 rpm to obtain seed solutions; 1 mL of the seed solution is transferred into 100 mL of LB liquid medium, added with 50 μL of 100 mg / mL AMP, and cultured at 37 °C and 200 rpm for about 3 h. When OD = 0.6, 10 mL of the bacterial solution is transferred into 1000 mL of LB liquid medium, added with 1000 μL of AMP, cultured at 37 °C for 4 h. When the bacterial solution becomes turbid to the logarithmic phase, 1 mM isopropyl-β-D-thiogalactoside (IPTG) is added, and protein expression is induced at 18 °C and 120 rpm for about 18 h; centrifuged at 4 °C and 3900 rpm, the supernatant is discarded, and the five recombinant bacteria are stored at -80 °C.

[0040] 3. Purification of recombinant proteins Resuspend the bacterial cells with lysis buffer containing 10 mM imidazole, sonicate on ice bath, and take the supernatant after centrifugation. Equilibrate the nickel column with washing buffer containing 20 mM imidazole, and then perform gradient elution with elution buffer containing different concentrations of imidazole to obtain the target proteins MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126, and verify the protein solubility and expression level by SDS-PAGE. Recombinant enterokinase (1 U:1 mg) was used to remove the fusion protein tag TrxA, and the protein size was verified by Tricine-SDS-PAGE. The results of Tricine-SDS-PAGE are as Figure 1 shown. From Figure 1 A, it can be obtained that the molecular weight of MmTX70 is consistent with the expected 4.3 kDa, the molecular weight of MmTX118 is consistent with the expected 7.1 kDa, the molecular weight of MmTX67 is consistent with the expected 5.4 kDa, the molecular weight of MmTX126 is consistent with the expected 9.0 kDa. From Figure 1 B, it can be obtained that the molecular weight of MmTX106 is consistent with the expected 7.7 kDa.

[0041] Example 2 This example provides the effects of the polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 in Example 1 on activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT).

[0042] The activated partial thromboplastin time (APTT) test mainly reflects the intrinsic coagulation pathway, and the length of APTT mainly depends on the activity of coagulation factors in the intrinsic coagulation pathway. The prothrombin time (PT) test is mainly used to evaluate the extrinsic coagulation pathway. If coagulation factors are deficient or their activity is reduced, PT will be prolonged. The principle of the thrombin time (TT) test is mainly based on adding a "standardized" thrombin solution to the tested plasma and observing the time required for plasma coagulation to reflect the time for fibrinogen in the plasma to be converted into fibrin, and to judge whether the fibrinogen in the plasma is abnormal or whether there are anticoagulant substances, etc.

[0043] In this example, the coagulation function tests (APTT, PT, TT) were performed using a kit according to the instructions of Beijing Zhongchi Weiye Technology Development Co., Ltd., and measured by an XL3200c automatic coagulation analyzer. The blank group was normal saline, and enoxaparin sodium was used as the positive control. The final concentration of all polypeptides was 25 μM. The results are as Figure 2 、 Figure 3 and Figure 4As shown in the figure, in the figure, "*", "**", "***", "****", and "ns" are used to represent the P-value ranges of different significance levels. "*" indicates that the P-value ≤ 0.05; "**" indicates that the P-value ≤ 0.01; "***" indicates that the P-value ≤ 0.001; "****" indicates that the P-value ≤ 0.0001; the more "*" there are, the more significant the result is. "ns" indicates that the P-value > 0.05, that is, the result is not significant.

[0044] From Figure 2 and Figure 3 it can be obtained that under the action of MmTX70, APTT is significantly reduced, and under the action of MmTX67 and MmTX106, APTT is significantly increased, while the PT test results are not significant. The above shows that MmTX70, MmTX67, and MmTX106 act on the intrinsic coagulation pathway rather than the extrinsic coagulation pathway, and MmTX70 has a procoagulant effect, while MmTX67 and MmTX106 have an anticoagulant effect. From Figure 4 it can be obtained that the thrombin time of MmTX67 and MmTX106 is significantly prolonged, indicating that MmTX67 and MmTX106 inhibit fibrin formation and have significant anticoagulant activity.

[0045] Example 3 This example provides the effects of the polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 in Example 1 on the recalcification time.

[0046] The recalcification experiment is to treat blood or plasma samples with anticoagulants to remove free calcium ions in the blood, so that the coagulation process cannot be normally initiated. When calcium ions are re-added, the coagulation factors are activated again, and the coagulation process restarts, so as to explore the effects of various factors on the coagulation process.

[0047] The specific steps are as follows: Dilute MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 to 125 μM or 250 μM; Pipette 20 μL of the sample into a 96-well plate and make 2 parallel samples; Add 50 μL of platelet-poor plasma (PPP), then incubate the 96-well plate in a shaker (37 °C, 200 rpm) for 10 min, add 30 μL of calcium ion solution at 37 °C, and immediately put it into an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 405 nm. The final concentrations of the samples are determined to be 25 μM and 50 μM respectively. The blank control group uses 20 μL of normal saline to replace 20 μL of the polypeptide sample; the negative control group uses 30 μL of normal saline to replace 30 μL of the calcium ion solution. The results are as Figure 5 and Figure 6 shown.

[0048] From Figure 5 and Figure 6It can be seen that the absorbance of the negative control group remained the lowest because no calcium ion solution was added, and the coagulation reaction was not initiated, so the absorbance was the lowest. The blank control group initiated the coagulation reaction after adding calcium ions, and the absorbance gradually increased with coagulation. MmTX70 could shorten the fibrin formation time at 25 μM, showing a procoagulant effect, while the effect was not obvious at 50 μM. MmTX67 and MmTX106 significantly inhibited fibrin formation at both 25 μM and 50 μM concentrations, showing a significant anticoagulant effect. The anticoagulant effect of MmTX67 and MmTX106 at 25 μM was better than that at 50 μM. The recalcification time of MmTX118 and MmTX126 was slightly prolonged at 50 μM concentration. The above results indicate that MmTX118, MmTX67, MmTX106, and MmTX126 all have an effect of prolonging the recalcification time. Among them, the anticoagulant effects of MmTX67 and MmTX106 are relatively significant.

[0049] Example 4 This example provides the effects of the polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 in Example 1 on thrombosis.

[0050] Thromboelastography (TEG) can dynamically monitor the entire blood coagulation process. TEG can well simulate the influence of the test sample on the in vivo real coagulation process by detecting and analyzing the strength and stability of the blood clot formed by the interaction between fibrin and platelets.

[0051] The specific steps are as follows: Take 32 μL of the diluted solutions of MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 (final concentration of 25 μM) respectively and mix them with 310 μL of platelet-poor plasma (PPP), then incubate at 37 °C for 5 min to obtain a mixture; turn on the thromboelastograph, adjust the interface and place the cup; add 20 μL of calcium ion solution to the cup, and then add 340 μL of the mixture, and operate the thromboelastograph for testing according to the instructions. To eliminate the influence of the solvent, a PBS (phosphate buffer solution) group and a DMSO (dimethyl sulfoxide) group were set as controls, and the results are as Figure 7 shown.

[0052] From Figure 7 it can be seen that MmTX70 shortened the blood clot formation time, showing procoagulant activity, MmTX118 slightly prolonged the blood clot formation time, showing antithrombotic activity, and the blood clot formation times of MmTX67, MmTX106, and MmTX126 were significantly prolonged, showing significant antithrombotic activity.

[0053] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polypeptide derived from Buthus martensii Karsch, characterized in that, The amino acid sequences of the polypeptides derived from Buthus martensii Karsch are shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO.

5.

2. A coding gene, characterized in that, It encodes the polypeptide derived from Buthus martensii Karsch as claimed in claim 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO.

10.

3. A recombinant plasmid, characterized in that, It is inserted with the encoding gene as claimed in claim 2.

4. A recombinant bacterium, characterized in that, It contains the recombinant plasmid as claimed in claim 3.

5. A procoagulant pharmaceutical composition, characterized in that, It includes a polypeptide derived from Buthus martensii Karsch with the amino acid sequence of SEQ ID NO.

1.

6. Use of the procoagulant pharmaceutical composition as claimed in claim 5 in the preparation of a procoagulant drug.

7. An anticoagulant pharmaceutical composition, characterized in that, It includes a polypeptide derived from Buthus martensii Karsch with the amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO.

5.

8. Use of the anticoagulant pharmaceutical composition as claimed in claim 7 in the preparation of an anticoagulant drug.

9. Use of a polypeptide derived from Buthus martensii Karsch with the amino acid sequence shown as SEQ ID NO. 1 in the preparation of a procoagulant drug.

10. Use of a polypeptide derived from Buthus martensii Karsch with the amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 in the preparation of an anticoagulant drug.

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