Coding gene of coral-derived anticoagulant polypeptide and application

Abstract

The invention provides a coding gene of coral-derived anticoagulant polypeptide and application, and belongs to the technical field of bioactive peptides. The invention provides an anticoagulant polypeptide GcKuz1. The anticoagulant polypeptide GcKuz1 comprises a mature peptide fragment as shown in SEQ ID No.1. The invention also provides a preparation method of the anticoagulant polypeptide GcKuz1. The polypeptide GcKuz1 is combined with KLKB1 in a thrombin system, so that the anti-thrombus and anti-coagulation effects are achieved, and the polypeptide does not cause hemolysis and cytotoxicity, has no bleeding risk and has good biological safety. The anticoagulant polypeptide disclosed by the invention has the effect of treating thrombotic diseases, such as arterial thrombotic diseases, venous thrombotic diseases and capillary thrombotic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to a coding gene of a coral-derived anticoagulant polypeptide and an application thereof. Background Art

[0002] Cardiovascular and cerebrovascular diseases are a general term for cardiovascular and cerebrovascular diseases. Their pathogenesis is complex and diverse, including ischemic or hemorrhagic diseases of the heart, brain and systemic tissues caused by risk factors such as hyperlipidemia, blood viscosity, atherosclerosis, and hypertension. Cardiovascular and cerebrovascular diseases have seriously threatened human health and quality of life, and have placed a huge burden on the social economy. Therefore, research on drugs to treat cardiovascular and cerebrovascular diseases has a huge social demand and important social significance.

[0003] Under normal physiological conditions, the body's hemostasis, anticoagulation and fibrinolysis systems interact and restrict each other and are regulated by neurohumoral factors, so that the blood will neither overflow the blood vessel wall and bleed, nor coagulate in the blood vessel and cause thrombosis. However, under pathological conditions, hyperprocoagulant function, anticoagulation or reduced fibrinolysis can lead to a prethrombotic state or thrombosis. Conversely, it can induce a hypocoagulable state or bleeding. The coagulation system is closely related to thrombotic diseases. Thrombosis is the most terrible complication of cardiovascular disease and the main cause of death in humans worldwide. Platelets and coagulation cascades are effective targets for antithrombotic therapy, but they have an inherent risk of bleeding.

[0004] Bioactive peptides are a class of short peptides or polypeptide molecules with specific biological activities. They regulate cell signal transduction, promote physiological reactions, and affect the physiological functions of organisms by binding to specific receptors. Animal-derived bioactive peptides have been a hot topic in the development of new drugs in recent years. However, due to factors such as strong toxicity and bleeding risks, there are very few polypeptide drugs for cardiovascular diseases used in clinical practice, which is far from meeting the growing demand of the anti-cardiovascular drug market. Therefore, how to obtain a highly active and low-toxic polypeptide drug for the prevention and treatment of cardiovascular diseases to meet the needs of a large number of cardiovascular disease drugs is an urgent problem to be solved. Summary of the invention

[0005] The present invention provides a gene encoding a coral-derived anticoagulant polypeptide and its application. The anticoagulant polypeptide has the ability to specifically bind to plasma kallikrein and has no bleeding risk, and is useful for treating thrombotic diseases.

[0006] The present invention provides an anticoagulant polypeptide, which comprises a mature peptide segment shown in SEQ ID No.1.

[0007] In one embodiment of the present invention, the anticoagulant polypeptide comprises the amino acid sequence shown in SEQ ID No.2.

[0008] The present invention provides a gene encoding the anticoagulant polypeptide.

[0009] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the mature peptide segment of the anticoagulant polypeptide is shown as SEQ ID No.3.

[0010] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the anticoagulant polypeptide is shown as SEQ ID No.4.

[0011] The present invention provides the use of the anticoagulant polypeptide in preparing a biomaterial having anticoagulant and / or antithrombotic activity.

[0012] In one embodiment of the present invention, the biological material comprises a drug.

[0013] The present invention provides a drug with anticoagulant and / or antithrombotic activity, wherein the active ingredient comprises the anticoagulant polypeptide and pharmaceutically acceptable auxiliary materials.

[0014] In one embodiment of the present invention, the dosage of the drug, calculated as anticoagulant polypeptide, is not less than 2 mg / kg mouse.

[0015] Beneficial effects: The present invention provides an anticoagulant polypeptide GcKuz1, which includes a mature peptide segment shown in SEQ ID No. 1, and is a novel Kunitz-type polypeptide predicted from the transcriptome of reef-building corals. The anticoagulant polypeptide takes plasma kallikrein in the coagulation system as a target. The anticoagulant polypeptide binds to KLKB1, thereby exerting antithrombotic and anticoagulant effects. The polypeptide does not cause hemolysis and cytotoxicity, has no risk of bleeding, and has good biosafety. The GcKuz1 polypeptide of the present invention has anticoagulant activity in vitro, specifically, GcKuz1 concentration-dependently prolongs the fibrin formation time in plasma recalcification and the activated partial thromboplastin time, but has no effect on the prothrombin time; the polypeptide GcKuz1 can concentration-dependently prolong the carotid artery thrombosis time induced by FeCl3 in mice; the polypeptide GcKuz1 can concentration-dependently inhibit carrageenan-induced tail thrombosis in mice; the polypeptide GcKuz1 can concentration-dependently inhibit and reduce the damage caused by stroke reperfusion, including reducing the ischemic area of ​​the mouse brain, improving the grip strength of the mouse and improving the neurobehavioral phenotype of the mouse. The anticoagulant polypeptide of the present invention has the effect of treating thrombotic diseases, such as arterial thrombotic diseases, venous thrombotic diseases and capillary thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The sequence, base sequence and multiple sequence alignment analysis results of the GcKuz1 polypeptide are shown in Figure A, in which A represents the full-length cDNA and amino acid sequence of GcKuz1, the green background represents the signal peptide sequence, and the red represents the mature peptide sequence; B represents the multiple sequence alignment of the GcKuz1 polypeptide and Kunitz-type polypeptides from different species;

[0017] Figure 2 The results of enzyme kinetics detection and SPR analysis to verify the binding and interaction between GcKuz1 and PlasmaKallikrein are shown in Figure 1. A represents the results of enzyme kinetics detection; B represents the results of surface plasmon resonance (SPR) analysis;

[0018] Figure 3 The figure is the result of the in vitro anticoagulant activity verification of GcKuz1 polypeptide, in which A and D represent PRT analysis; B and E represent APTT analysis; C and F represent PT analysis; the data represent the mean ± standard error of three independent experiments (n>=3); **p<0.001, ***p<0.001 compared with the control group;

[0019] Figure 4 The figure shows the therapeutic effect of GcKuz1 on the ferric chloride-induced mouse carotid artery thrombosis experiment; A in the figure shows the imaging result of the blood flow in the mouse carotid artery; B shows the carotid artery embolization time of the mouse; C shows the ROI value analysis result of the blood flow in the mouse carotid artery; the data are the mean ± standard error (n = 6); ****p < 0.0001 compared with the control group;

[0020] Figure 5 The figure shows the therapeutic effect of GcKuz1 on carrageenan-induced mouse tail thrombosis experiment; A in the figure shows a representative picture of mouse tail thrombosis; B shows the length of mouse tail thrombosis; data represent mean ± standard error (n = 6); ****p < 0.0001 compared with the model group;

[0021] Figure 6 The figure shows the therapeutic effect of GcKuz1 on cerebral ischemia-reperfusion injury in tMCAO mouse model; A and B in the figure show TTC staining and statistical graphs of brain sections of tMCAO mice; C shows the nerve injury scores of mice in different treatment groups; D shows the grip strength of mice in different treatment groups; data represent mean ± standard error (n = 6); *p < 0.05, ***p < 0.001, ****p < 0.0001 compared with the control group;

[0022] Figure 7The results of the test that GcKuz1 does not cause bleeding, hemolysis risk and cytotoxicity. A is the hemolytic activity of GcKuz1 red blood cells, in which TritonX-100 is a positive drug; B is the evaluation of the bleeding risk of GcKuz1 using a mouse tail bleeding test (heparin sodium is a positive drug); 24 hours after administration of GcKuz1, CCK8 analysis results of SH-SY5Y cells (C), mouse spleen cells (D) and mouse peritoneal macrophages (E) cell viability; data are from three independent experiments, and the data represent the mean ± standard error (n = 3-5); #### p<0.0001, ****p<0.0001, NS indicates no significant difference compared with the control group. DETAILED DESCRIPTION

[0023] The present invention provides an anticoagulant polypeptide, which comprises a mature peptide segment shown in SEQ ID No.1.

[0024] The anticoagulant polypeptide of the present invention is derived from reef-building coral and is named GcKuz1. The mature peptide segment of GcKuz1 has three pairs of disulfide bonds, a molecular weight of 6095.83 Daltons, and an amino acid sequence as shown in SEQ ID No. 1: SICHLPKDVGFCKALIRRYFYNTSKGKCEMFHYGGCYGNKNNFKTLVSCKRAC. The complete primary structure sequence of the polypeptide GcKuz1 of the present invention is shown in SEQ ID No. 2: MKSTFLVLVILLYYHAVQASICHLPKDVGFCKALIRRYFYNTSKGKCEMFHYGGCYGNKNNFKTLVSCKRACERNKRSCPRRETTSGQ.

[0025] The present invention provides a gene encoding the anticoagulant polypeptide.

[0026] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the mature peptide segment of the anticoagulant polypeptide is as shown in SEQ ID No.3: TCAATCTGCCACCTACCTAAAGATGTCGGATTCTGCAAGGCCCTCATTCGCCGGTATTTTTACAACACTTCCAAGGGGAAATGTGAGATGTTTCACTACGGTGGATGCTATGGTAATAAAAATAACTTCAAGACACTTGTGAGTTGCAAGAGAGCGTGC; and the nucleotide sequence of the gene encoding the anticoagulant polypeptide is as shown in SEQ ID Shown in No.4: 5'-ATGAAGTCCACCTTCCTAGTTTTGGTTATATTGTTGTACTATCACGCAGTTCAGGCTTCAATCTGCCACCTACCTAAAGATGTCGGATTCTGCAAGGCCCTCATTCGCCGGTATTTTTACAACACTTCC AAGGGGAAATGTGAGATGTTTCACTACGGTGGATGCTATGGTAATAAAAATAACTTCAAGACACTTGTGAGTTGCAAGAGAGCGTGCGAACGTAACAAGCGGTCTTGCCCTCGACGGGAGACAACATCTGGTCAA-3'.

[0027] The present invention provides the use of the anticoagulant polypeptide in preparing a biomaterial having anticoagulant and / or antithrombotic activity.

[0028] In the embodiment of the present invention, enzyme kinetics and surface plasmon resonance methods are used to confirm that the polypeptide GcKuz1 can bind to Plasmakallikrein / KLKB1, and the KD constant of the binding is 8.63 μM; at the same time, in vitro experiments are used to confirm that the polypeptide GcKuz1 concentration-dependently prolongs the fibrin formation time in plasma recalcification and the activated partial thromboplastin time, but has no effect on the prothrombin time. The present invention confirms through in vivo experiments that the polypeptide GcKuz1 can concentration-dependently prolong the time of FeCl3-induced carotid artery thrombosis in mice, and the effective dose is not less than 2 mg / kg, and the polypeptide GcKuz1 can concentration-dependently inhibit carrageenan-induced mouse tail thrombosis; the polypeptide GcKuz1 can concentration-dependently inhibit and reduce the damage caused by stroke reperfusion, including reducing the ischemic area of ​​the mouse brain, improving the grip strength of the mouse and improving the neurobehavioral phenotype of the mouse. The polypeptide GcKuz1 does not cause obvious hemolysis and bleeding and has no cytotoxicity, and has high biosafety. In summary, the polypeptide GcKuz1 of the present invention has potential application value in the treatment of thrombotic diseases.

[0029] The present invention provides a drug with anticoagulant and / or antithrombotic activity, wherein the active ingredient comprises the anticoagulant polypeptide and pharmaceutically acceptable auxiliary materials.

[0030] In one embodiment of the present invention, the dosage of the drug, calculated as anticoagulant polypeptide, is not less than 2 mg / kg mouse.

[0031] To further illustrate the present invention, the coding gene and application of a coral-derived anticoagulant polypeptide provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Discovery of GcKuz1 polypeptide sequence and its encoding gene

[0033] Through Blast annotation and Pfam analysis, a transcript fragment (ID: c102034_g1_i1) with a typical Kunitz domain was identified from the transcript sequence of Goniopora columna. Through multiple sequence alignment analysis with Kunitz polypeptide homologs from different species, it was confirmed that GcKuz1 is a new type of GcKuz1 polypeptide derived from coral.

[0034] The results are as follows Figure 1 As shown, GcKuz1 is a novel coral-derived Kunitz polypeptide having a mature peptide sequence as shown in SEQ ID No.1 and a complete amino acid sequence as shown in SEQ ID No.2.

[0035] Example 2 Determination of the target of GcKuz1 polypeptide

[0036] 1. Enzyme kinetics detection:

[0037] 110 nM KLKB1 and different final concentrations of GcKuz1 peptide (2-16 μM) were added to 50 μL PBS buffer and incubated at room temperature for 5 min. Then, 50 μL of substrate Chromogenix S-2302 was added. TM (S820340, Chromogenix, Ita), and then the absorbance was continuously measured at 450 nm using an ELISA reader within 30 min. Figure 2 As shown in A, GcKuz1 peptide can inhibit the activity of PlasmaKallikrein.

[0038] 2. Surface Plasmon Resonance (SPR):

[0039] A CM5 sensor chip was activated for 20 minutes at a flow rate of 5 μL / min using 0.4M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide. The KLKB1 protein was diluted to 20 μg / mL with 1 mL sodium acetate (10 mM, pH 5.0) and flowed over the activated surface to achieve the target response value (RU). The remaining activation sites on the chip were blocked with 45 μL of hexanolamine (1 M, pH 8.5). At a flow rate of 30 μL / min, a Biacore S200 instrument (USA) was used to record real-time detection results. Subsequently, to determine the equilibrium dissociation constant (KD) of GcKuz1 and KLKB1, serially diluted GcKuz1 (10, 5, 2.5 and 1.25 μM) was treated in 150 mM phosphate buffered saline, pH 7.4, and its interaction with KLKB1 immobilized on the chip surface was analyzed using the BIAevaluation program.

[0040] The surface plasmon resonance (SPR) analysis results of the interaction between GcKuz1 and Plasma Kallikrein are shown in Figure 2 As shown in B, GcKuz1 can bind to KLKB1, and the KD constant of the binding is 8.63 μM.

[0041] Example 3 GcKuz1 polypeptide has anticoagulant activity in vitro

[0042] In this example, the anticoagulant activity of the polypeptide GcKuz1 provided by the present invention was detected by plasma recalcification time (PRT), activated partial thromboplastin time (APTT) and prothrombin time (PT). The polypeptide GcKuz1 described in this example was commissioned by Gutuo Biotechnology Company through chemical solid phase synthesis.

[0043] 1. Plasma recalcification time (PRT) determination: In a 96-well plate, 19 μL of healthy human plasma, 1 μL of different final concentrations of GcKuz1 polypeptide (2 μM, 4 μM, 8 μM, 16 μM) and 40 μL of HEPES buffer (150 mM Hepes, 20 mM NaCl, pH 7.4) were added to each well and incubated at 37°C for 5 min. After the incubation, 40 μL of CaCl2 solution preheated at 37°C for 10 min was added to each well, and the mixture was immediately mixed. The absorbance was continuously detected at a wavelength of 650 nm within 30 min using an enzyme reader, and the fibrin formation time was recorded.

[0044] 2. Activated partial thromboplastin time (APTT) assay: According to the instructions of the activated partial thromboplastin time (APTT) kit (TC0306100T, LEAGENE, China), 49 μL of healthy human plasma, 1 μL of different final concentrations of GcKuz1 polypeptide (2 μM, 4 μM, 8 μM, 16 μM) and 50 μL of APTT ellagic acid solution were incubated at 37°C for 5 min. After the incubation, 50 μL of CaCl2 solution preheated at 37°C for 10 min was added, and the mixture was immediately mixed. The absorbance was continuously detected at a wavelength of 650 nm within 3 min using an enzyme marker, and the fibrin formation time was recorded.

[0045] 3. Prothrombin time (PT) determination: According to the instructions of PTASSAYKIT (GMS10176, GENMED, USA), 49 μL of healthy human plasma and 1 μL of different final concentrations of GcKuz1 peptide (2 μM, 4 μM, 8 μM, 16 μM) were incubated at 37°C for 5 min. After the incubation, 50 μL of preheated PT reagent was immediately added to detect its absorbance within 3 min, and the fibrin formation time was recorded.

[0046] The results are as follows Figure 3 As shown, GcKuz1 concentration-dependently prolonged the fibrin formation time in plasma recalcification and the activated partial thromboplastin time, but had no effect on the prothrombin time.

[0047] Example 4 GcKuz1 polypeptide inhibits ferric chloride-induced carotid artery thrombosis in mice

[0048] In this example, the GcKuz1 polypeptide provided by the present invention has an inhibitory effect on ferric chloride-induced carotid artery thrombosis in mice.

[0049] Six-week-old C57 BL / 6J male mice (about 20 g) were used in the experiment and divided into 6 groups, namely, saline group, GcKuz1 peptide treatment group (1 mg / kg, 2 mg / kg and 4 mg / kg) and positive drug heparin sodium group (2500 U / kg). 10 minutes after the above drugs were injected into the tail vein of mice, 1.5% sodium pentobarbital (40 mg / kg) was intraperitoneally anesthetized, fixed in supine position, the neck was disinfected with 75% alcohol, a midline incision was made in the neck, the mouse skin and subcutaneous tissue were cut layer by layer, the sternocleidomastoid muscle was separated, the anterior belly of the digastric muscle was cut off, and the left common carotid artery (CCA) of the mouse was exposed. A filter paper was punched with a 5 mm diameter puncher, and a filter paper soaked in saline was placed on the lower side of the common carotid artery to remove background interference. A filter paper soaked in 10% FeCl3 was covered on the upper side of the artery. After FeCl3 stimulation, the filter paper was removed and the residual FeCl3 was washed off with saline. Then the mice were placed under a laser speckle flowmeter to observe blood flow changes and record vascular flow values ​​at 5 minutes and 10 minutes to evaluate the effect of GcKuz1 polypeptide on the time of carotid artery thrombosis in mice.

[0050] The results are as follows Figure 4 As shown, the polypeptide GcKuz1 can prolong the time of FeCl3-induced carotid artery thrombosis in mice in a concentration-dependent manner, and the effective doses are 2 mg / kg and 4 mg / kg.

[0051] Example 5 GcKuz1 polypeptide inhibits carrageenan-induced mouse tail thrombosis

[0052] In this example, the GcKuz1 polypeptide provided by the present invention has an inhibitory effect on carrageenan-induced mouse tail thrombosis.

[0053] Male 6-week-old Balb / C mice were intraperitoneally injected with 1% carrageenan (type I) (CC3171, coolaber, China) for 30 minutes, and then injected with normal saline, different concentrations of GcKuz1 peptide (1 mg / kg, 2 mg / kg and 4 mg / kg) and positive drug sodium heparin (2500 U / kg) through the tail vein. The length of the tail thrombus of the mice was observed and recorded 24 hours later to evaluate the effect of GcKuz1 peptide on the formation of tail thrombosis in mice.

[0054] The results of the therapeutic effect of GcKuz1 on carrageenan-induced mouse tail thrombosis experiment are as follows Figure 5 As shown, the polypeptide GcKuz1 can inhibit carrageenan-induced mouse tail thrombosis in a concentration-dependent manner, and the effective doses are 2 mg / kg and 4 mg / kg.

[0055] Example 6 GcKuz1 polypeptide alleviates damage caused by cerebral stroke reperfusion in mice

[0056] Six-week-old male C57 BL / 6J mice (about 20 g) were anesthetized with 1.5% sodium pentobarbital (40 mg / kg). After moderate to deep anesthesia, the rat skin and subcutaneous tissue were cut layer by layer through a midline incision in the neck, the sternocleidomastoid muscle was separated, the anterior belly of the digastric muscle was cut, and the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were exposed. The thyroid artery and pharyngeal artery on the ECA were coagulated and cut using an electrocoagulator. The distal end of the ECA was ligated, and the line was hung on its proximal end to temporarily clamp the CCA and ICA. The ECA was cut, and the line plug was inserted from the ECA stump into the ICA, the ECA stump was ligated, the artery clamp of the ICA was removed, and the line was slowly pushed from the ICA to the inside and above. The direction was adjusted appropriately, inserted to the line plug mark, and the timing was started. After 60 minutes, the line plug was removed, and the incision was sutured after observing no active bleeding. The whole process was kept warm with an electric blanket at 36-37°C. After awakening, the mice were returned to the cage and allowed to eat freely. At 24 hours after stroke, the neurological deficit score and muscle strength test were evaluated and recorded. The brain tissue was then removed and placed in a brain mold (on ice), cut into 2 mm thick slices along the optic chiasm, placed in 2% TTC phosphate buffer, stained in a 37°C incubator for 30 minutes, and then fixed with 4% paraformaldehyde overnight and photographed.

[0057] Ischemia volume percentage = [ischemic volume - (left hemisphere volume - right hemisphere volume)] / right hemisphere volume × 100. Drugs were administered via tail vein 10 min before ischemia-reperfusion. The experimental groups included normal saline group, different concentrations of GcKuz1 peptide (1 mg / kg, 2 mg / kg and 4 mg / kg) and positive drug ecallantide group.

[0058] The therapeutic effect of GcKuz1 on cerebral ischemia-reperfusion injury in tMCAO mouse model Figure 6 As shown, the peptide GcKuz1 can inhibit and alleviate the damage caused by stroke reperfusion in a concentration-dependent manner, including reducing the ischemic area of ​​the mouse brain, improving the grip strength of the mouse and improving the neurobehavioral phenotype of the mouse. The effective dose is 2 mg / kg and 4 mg / kg.

[0059] Example 7 GcKuz1 polypeptide has no cytotoxicity, hemolysis and bleeding risks

[0060] 1. Cell hemolysis experiment

[0061] Gently pour the red blood cells into 50 mL, add saline along the wall, centrifuge at 3500 rpm for 10 min at room temperature, and repeat this step 3 times. Add 20 mL of saline for the last time to adjust the final blood cell concentration to 1×10 8100 μL of GcKuz1 (6.25, 12.5, 25 and 50 μM) was added to each well, and then 100 μL of blood cells was added to each well, followed by incubation at 37°C for 30 min, centrifugation at 3500 rpm for 10 min at room temperature, and 150 μL of supernatant was taken for detection at an absorbance of 540 nm.

[0062] The hemolytic activity of GcKuz1 on erythrocytes Figure 7 As shown in A, the results of the red blood cell hemolysis experiment show that there is no significant difference in the absorbance of 0-25 μM GcKuz1 at 540 nm, indicating that GcKuz1 will not damage blood cells. Since GcKuz1 is administered through the tail vein, GcKuz1 first enters the blood, so it is a very important factor to first determine whether GcKuz1 has a destructive effect on red blood cells.

[0063] 2. Cytotoxicity assay

[0064] 100 μL of human neuroblastoma cells (SH-SY5Y), mouse peritoneal macrophages and mouse spleen cells were added at 10 4 The cells were seeded at a density of 100 μg / mL in a 96-well plate. After culturing the cells at 5% CO2 and 37°C for 24 hours, different final concentrations of GcKuz1 (3.125-50 μM) were added and incubated for 24 hours. Then, 10 μL Cell Counting Kit-8 was added and incubated for 90 minutes; the absorbance at 450 nm was measured.

[0065] This experiment was conducted to evaluate the safety of the GcKuz1 peptide. The results showed that GcKuz1 had no cytotoxicity (see Figure 7 (CE).

[0066] 3. Mouse tail bleeding experiment

[0067] Ten minutes after the tail vein injection of GcKuz1 (1, 2, and 4 mg / kg) and sodium heparin (positive drug), the mouse tail was disinfected with anhydrous ethanol, a mark was made with a pen at 0.2 mm from the tip of the tail, and a 2 mm long tail of a 6-week-old C57 BL / 6J mouse was cut with a professional scalpel (sterilized), and then placed in 37°C saline. The bleeding time of the mouse tail was recorded to assess whether GcKuz had a bleeding risk.

[0068] Results Figure 7 Middle B. The tail bleeding time of mice was used to determine whether GcKuz1 has a bleeding risk. The results showed that GcKuz1 does not have a bleeding risk.

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An anticoagulant polypeptide, characterized in that: The anticoagulant polypeptide includes the mature peptide segment shown in SEQ ID No.

1.

2. The anticoagulant polypeptide according to claim 1, characterized in that: The anticoagulant polypeptide comprises the amino acid sequence shown in SEQ ID No.

2.

3. A gene encoding the anticoagulant polypeptide according to claim 1 or 2.

4. The gene according to claim 3, characterized in that The nucleotide sequence of the gene encoding the mature peptide segment of the anticoagulant polypeptide is shown in SEQ ID No.

3.

5. The gene according to claim 3, characterized in that The nucleotide sequence of the gene encoding the anticoagulant polypeptide is shown in SEQ ID No.

4.

6. Use of the anticoagulant polypeptide according to claim 1 or 2 in the preparation of a biomaterial having anticoagulant and / or antithrombotic activity.

7. The use according to claim 6, characterized in that: The biological material includes a drug.

8. A drug having anticoagulant and / or antithrombotic activity, characterized in that: The active ingredient comprises the anticoagulant polypeptide according to claim 1 or 2, and also comprises pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that: The dosage of the drug, calculated as anticoagulant polypeptide, is not less than 2 mg / kg mouse.

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