A snake venom thrombin-like protein and its preparation method and application

Abstract

The present invention discloses a snake venom thrombin protein and its preparation method and application. It belongs to the technical field of snake venom protein. The present invention uses SUPERDEX 75INCREASE 10 / 300GL gel chromatography column and C18SP‑100‑5‑ODS‑P liquid chromatography column to separate and purify a protein 3185 with thrombin activity from the venom of Agkistrodon acutus. Protein spectrum identification combined with transcript data analysis shows that the number of peptide segments of the protein is 10, the number of amino acids is 260, the protein molecular weight is 29.1KDa, and the isoelectric point is 8.18. The enzyme activity method measures the enzyme specific activity to be 1032U / mg. The protein reacts with the thrombin-specific chromogenic substrate S‑2238, and the reaction conforms to enzyme kinetics. The protein only acts on the Aα chain of fibrinogen, releases fibrin polypeptide A to form a loosely structured fibrinogen monomer, and forms a soluble fibrin degradation product.

Description

Technical Field

[0001] The present invention relates to the technical field of snake venom proteins, and more particularly to a snake venom thrombin-like protein and a preparation method and application thereof. Background Art

[0002] The sharp-nosed pit viper (Deinagkistrodon) belongs to the Viperidae subfamily, also known as Agkistrodon acutus, Five-step snake, and Hundred-step snake. As a terrestrial wild animal that is beneficial to the ecology and has important economic and scientific research value, the sharp-nosed pit viper is protected at the national level and listed as a provincial key protected animal. It is also one of the common Chinese medicinal materials. Wild snake resources are decreasing day by day, and the sharp-nosed pit viper is listed as an endangered animal. At present, most of them are artificially bred. It is a snake with important economic and medicinal value.

[0003] According to statistics, there are more than 3,150 species of snakes in the world, of which 20% are venomous snakes. Venomous snake bites have high mortality and disability rates. It is estimated that 270,000 people are bitten by venomous snakes every year, 100,000 die from venomous snake bites, and 400,000 people are left with serious sequelae. The venom glands under the skin of the snake's cheeks are the main difference between venomous snakes and non-venomous snakes. The venom secreted by the venom glands plays an important role in the predation, attack and defense of venomous snakes. When a snake bites a mammal, the proteins and peptides in the venom will cause disease if they are not processed. The diversity of snake venom components provides medicinal chemists with a series of highly specific and biologically active compounds, which have great potential as the main compounds of new therapeutic drugs. Studying the structure and function of a single toxin has been the mainstream direction of snake venom research in recent decades, and the development of biological activity of snake venom peptides is a hot spot in the field of new drug research and development.

[0004] The composition of venom is affected by environmental factors, climate, age, gender and other factors, resulting in great differences in snake venom components between and within species. Depending on the type of snake, its main physiological activity is also different, mainly covering the impact on the nervous system, the effect on muscle tissue, the ability to damage cells, and the toxic effects on the blood circulation system, including neurotoxicity, myotoxicity, cytotoxicity, hemotoxicity, etc. The diversity of snake venom composition is a huge challenge for the clinical treatment of venomous snake bites. The variability of snake venom components limits the supply and large-scale production of anti-venom serum, showing its disadvantages.

[0005] In summary, how to provide a snake venom polypeptide is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a snake venom thrombin-like protein and a preparation method and application thereof.

[0007] The present invention extracts and purifies a new type of anticoagulant based on the venom of Agkistrodon acutus, and preliminarily completes its activity determination.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A snake venom thrombin-like protein, the amino acid sequence of which is as follows:

[0010] MVLIRVLANLLILQLSYAQKSSELIIGGDECDINEHRFLVGLYTSRSRRF

[0011] YCCGTLINPEWVVTAAHCERKNIRIKLGMHSKNTPNEDVQIRVPKEKFFCL

[0012] SSKTYTKWSNDIMLIRLKRPVNNSTHIAPVSLPSNPPSLGSVCRIMGWGTIT

[0013] SPKKTYPDVPHCANILDYEVCREAHPWLPATSRTWCAGILEGGKDTCG

[0014] GDSGGPLICDGQFQGIVSWGWNPCAQQREPGHYTKVIDYNDWIQRNIAGN

[0015] TDATCPP*, SEQ ID NO.1.

[0016] Further, the following peptides are included:

[0017] EAHPWLPATSR, SEQ ID NO. 2;

[0018] IMGWGTITSPK, SEQ ID NO. 3;

[0019] KTYPDVPHCANILDYEVCR,SEQ ID NO.4;

[0020] NTPNEDVQIRVPK, SEQ ID NO.5;

[0021] TWCAGILEGGK, SEQ ID NO. 6;

[0022] TYPDVPHCANINILDYEVCR,SEQ ID NO.7;

[0023] VIDYNDWIQR, SEQ ID NO. 8;

[0024] WSNDIMLIR;

[0025] FFCLSSK;

[0026] FLVGLYTSR.

[0027] The preparation method of the snake venom thrombin-like protein is to separate and purify the snake venom freeze-dried powder through a SUPERDEX 75INCREASE10 / 300GL gel chromatography column, a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, a C18 SP-100-5-ODS-P liquid chromatography column, and a C18 SP-100-5-ODS-P liquid chromatography column in sequence.

[0028] Further, the following steps are included:

[0029] (1) The lyophilized snake venom powder was isocratically eluted through a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, with the eluent being a PBS solution, and the fraction with the highest thrombin activity was collected;

[0030] (2) isocratically eluting the fraction obtained in step (1) through a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, using a PBS solution as the eluent, and collecting the fraction with the highest thrombin activity;

[0031] (3) gradient eluting the components obtained in step (2) through a C18 SP-100-5-ODS-P liquid chromatography column with a mobile phase system of water and acetonitrile, and collecting the components with the highest thrombin activity;

[0032] (4) The components obtained in step (3) are subjected to gradient elution through a C18 SP-100-5-ODS-P liquid chromatography column with a mobile phase system of water and acetonitrile, and the components with the highest thrombin activity are collected.

[0033] Furthermore, the elution flow rate of steps (1) and (2) is 0.8 mL / min.

[0034] Furthermore, the elution flow rate of steps (3) and (4) is 2 mL / min.

[0035] Application of the above-mentioned snake venom thrombin-like protein in the preparation of anticoagulant drugs.

[0036] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0037] The present invention uses a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column and a C18SP-100-5-ODS-P liquid chromatography column to separate and purify a protein 3185 with thrombin activity from the venom of Agkistrodon acutus. Protein spectrum identification combined with transcript data analysis shows that the number of peptide segments of the protein is 10, the number of amino acids is 260, the protein molecular weight is 29.1KDa, and the isoelectric point is 8.18. The enzyme activity method measures the enzyme specific activity to be 1032U / mg. The protein reacts with the thrombin-specific chromogenic substrate S-2238, and the reaction conforms to the enzyme kinetics. The protein only acts on the Aα chain of fibrinogen, releases fibrin polypeptide A to form a loosely structured fibrinogen monomer, and forms a soluble fibrin degradation product. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0039] Figure 1 The standard curve of enzyme activity in Example 1 of the present invention;

[0040] Figure 2 This is the elution diagram of the SUPERDEX 75INCREASE 10 / 300GL gel chromatography column of the snake venom mother solution in Example 2 of the present invention;

[0041] Figure 3 This is an SDS-PAGE image of component 1 in Example 2 of the present invention, wherein the lanes from left to right are component 1 and Maker protein;

[0042] Figure 4 This is the elution profile of SUPERDEX 75INCREASE 10 / 300GL gel chromatography column of component 1 in Example 2 of the present invention;

[0043] Figure 5 This is the elution profile of the C18 SP-100-5-ODS-P liquid chromatography column of components 1-3 in Example 2 of the present invention;

[0044] Figure 6 This is the elution profile of the C18 SP-100-5-ODS-P liquid chromatography column of component b in Example 2 of the present invention;

[0045] Figure 7 This is the spectrum of the purified protein in Example 3 of the present invention;

[0046] Figure 8This is the hydrolysis reaction curve of the enzyme and the chromogenic substrate S-2238 in Example 4 of the present invention;

[0047] Fig. 9 This is the SDS-PAGE electrophoresis diagram of the hydrolysis of fibrinogen by the purified protein 3185 in Example 4 of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Instruments and Materials

[0050] Quintix224-1CN 1 / 10,000 electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0051] YP-B5002 1% electronic balance (Shanghai Guangzheng Medical Instruments and Equipment Co., Ltd.);

[0052] SB-5200D ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.);

[0053] Microplate reader (PERKINELMER EnVision2105);

[0054] Electrophoresis apparatus (Bio-Rad);

[0055] Electrophoresis tank (Bio-Rad);

[0056] Preparative chromatography (Beijing Aonuo Technology Co., Ltd.);

[0057] Water Purifier (Thermo Scientific);

[0058] Mass spectrometer (Thermo Scientific);

[0059] Chromatography system (Easy-nLC1200, Thermo Scientific);

[0060] Centrifuge (Eppendorf).

[0061] Trifluoroacetic acid (McLean T875598);

[0062] Fibrinogen (China Food and Drug Inspection Institute 140607-202143);

[0063] Defibrase (China Institute for the Authentication of Pharmaceutical and Biological Products 140620-200802);

[0064] S-2238 (Source Leaf Biotechnology S27564);

[0065] BCA protein kit (Elarerite);

[0066] PageRuler TM prestained protein molecular weight standard (Thermo);

[0067] SDS-PAGE protein loading buffer (5X) (Biyuntian);

[0068] Precast gel (YaSe).

[0069] Acetonitrile was of chromatography grade, and sodium chloride, Tris, and hydrochloric acid were of analytical grade.

[0070] The preparation method of the freeze-dried powder of Agkistrodon acutus venom is as follows: using the utility model patent ZL201420548959.4 "A step-type snake feeding machine with a venom extraction device", holding the snake's neck with anti-needle gloves, first let the Agkistrodon acutus open its mouth naturally and bite the membrane of the venom extractor to excrete venom naturally. Collect the venom, store it below -40°C, freeze-dry it in a low-temperature freeze dryer for 20 hours to a water content of less than 5%, and store it at -20°C.

[0071] The Agkistrodon acutus was collected from Hunan Yongzhou Yishe Co., Ltd. In September 2020, the National Forestry and Grassland Administration issued the "Notice on Standardizing the Classification and Management Scope of Wild Animals Banned from Eating", which clearly prohibits the breeding of Agkistrodon acutus for the purpose of eating, while allowing breeding for non-edible fields such as medicine, exhibition and scientific research.

[0072] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0073] Example 1

[0074] Evaluation methods for screening active components

[0075] Taking thrombin activity as the target of snake venom purification, the enzyme activity was evaluated according to the titer determination method of defibrase in the national drug standard. Adaptive adjustments were made according to the actual situation, as follows:

[0076] Preparation of Tris buffer (pH 7.4): Dissolve 2.42g Tris and 0.585g NaCl in appropriate amount of water. Adjust pH to 7.4 with 1mol / L hydrochloric acid solution and add water to make up to 500mL. Take defibrase and prepare standard solutions containing 0.25U, 0.5U, 1.0U, and 2.0U defibrase per mL with Tris buffer, and draw a standard curve.

[0077] Take 200μL of 0.4% coagulable protein solution, incubate in a 37℃ water bath for 2min, add 200μL of standard solution, shake well and start timing immediately, observe the initial coagulation time in the water bath, measure 5 times for each concentration, and take the average value (the difference between the 5 measurement results shall not be greater than 10% of the average value, otherwise it will be invalid). Calculate the regression equation of enzyme activity and initial coagulation time on double logarithmic coordinate paper. The test sample determination method is the same as above, and the enzyme activity is calculated by regression equation. BCA protein concentration is determined according to the kit method.

[0078] Defibrase converts soluble plasma fibrinogen into insoluble polymer fibrin. Plotting points on double logarithmic coordinate paper, the initial coagulation time of defibrase is linearly related to the activity of defibrase. The difference between the five measured data of the initial coagulation time of plasma fibrinogen with different defibrase activities is less than 10% of the average value. With the defibrase activity unit (U) as the horizontal axis and the time (S) as the vertical axis, a linear fit is performed on the double logarithmic coordinate paper. The standard curve is as follows: Figure 1 As shown, the linear regression equation is y = -0.9772x + 0.71425, R 2 is 0.99735. That is, log Y = -0.9772log X + 0.71425.

[0079] Example 2

[0080] Isolation and purification of Agkistrodon acutus venom

[0081] Dissolve 10g of snake venom freeze-dried powder in 100mL of pure water to prepare a uniform mother solution, package it in EP tubes, 2mL per tube, and store it at -20℃. Take an appropriate amount of snake venom mother solution, use preparative chromatography, SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, elute according to the molecular weight of the protein, the elution solution is PBS at pH 7.4, isocratic elution, flow rate is 0.8mL / min, sample volume is 50μL, and dual wavelength detection is 215nm and 280nm. Collect each component according to the peak for protein concentration determination and enzyme activity determination. Track the component with high thrombin activity, and continue to separate it using SUPERDEX 75INCREASE 10 / 300GL gel chromatography column according to the actual peak shape, and the elution conditions are the same as above. The protein was further separated and purified using a C18 SP-100-5-ODS-P liquid chromatography column and desalted. The mobile phase system was liquid A (water) and liquid B (acetonitrile). The gradient elution conditions were as shown in Table 1. The components were collected and the protein concentration and enzyme activity were determined. After the active components were determined, they were freeze-dried, weighed, and stored at -20°C.

[0082] Table 1C18 SP-100-5-ODS-P HPLC Column Elution Gradient

[0083]

[0084] According to the molecular weight of the protein, the snake venom mother solution was eluted using a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column. The elution results are as follows: Figure 2 As shown, we divided it into 7 components according to the peak type. According to the molecular weight of Thrombin of 37KDa and the instruction manual of SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, we roughly determined that the target protein was mainly concentrated in the first three components. We used the BCA protein concentration detection kit to detect the protein and perform enzyme activity assay on the first three components. The results showed that the concentrations of components 1, 2, and 3 were 0.58mg / mL, 0.21mg / mL, and 1.60mg / mL, respectively. The results of enzyme activity assay showed that the initial coagulation time of component 1 diluted 41 times at this concentration and reacting with coagulated protein was 6.76s, the initial coagulation time of component 2 after dilution 41 times was 199s, and component 3 did not show initial coagulation after only 7min of dilution 11 times. It can be seen that the activity is ranked from large to small as components 1, 2, and 3, and the enzyme activities of components 1 and 2 calculated by the regression equation are 272.09U and 23.91U, respectively. Since component 3 did not coagulate after more than 7 minutes, the detailed time was not recorded, so its enzyme activity was not calculated. At present, component 1 has been identified as the active component. SDS-PAGE electrophoresis analysis was performed on component 1. The electrophoresis diagram is shown in Figure 3 .

[0085] By peak type ( Figure 2 ) It can be seen that component 1 has a non-smooth curve at 215nm, and the electrophoresis diagram ( Figure 3 ) also showed that component 1 could be further separated and eluted according to molecular weight, so SUPERDEX 75INCREASE 10 / 300GL gel chromatography column was used again to elute and separate component 1. The elution results are shown in Figure 4 It was divided into three components, the distillate from 11min-11.5min was component 1-1, the distillate from 11.5min-12.5min was component 1-2, and the distillate from 12.5min-13min was component 1-3. The enzyme activity assay results showed that components 1-1 and 1-2 did not coagulate within 10min, and the enzyme activity of component 1-3 was 566.42U / mg, that is, component 1-3 had the highest enzyme activity.

[0086] Collect enough fractions 1-3 and use C18 SP-100-5-ODS-P liquid chromatography column to continue to separate and purify the protein and elute the salt. The elution results are shown in Figure 5Components a (39 min), b (47 min), and c (58 min) were collected, and the enzyme activities of the components were measured to be 2.027 U / mg for component a, 746.93 U / mg for component b, and 21.243 U / mg for component c, that is, the enzyme activities were ranked from high to low as component b, c, and a.

[0087] Component b was further purified using a C18 SP-100-5-ODS-P liquid chromatography column. The elution results are shown in Figure 6 . Collect the distillate of the 47min peak (with Figure 5 The corresponding peak b) was freeze-dried to obtain 5.53 mg of freeze-dried powder, which was stored at -20°C for subsequent physical and chemical analysis.

[0088] Example 3

[0089] Mass spectrometry identification of isolated and purified proteins

[0090] (1) Proteolysis

[0091] Take the purified sample and add an appropriate amount of SDT lysis buffer (4% SDS, 100mM Tris-HCl), add an appropriate amount of TCEP / CAA mixed solution to the sample, and reduce the protein in a boiling water bath at 100℃ for 5min. Add an appropriate amount of UA buffer (8M Urea, 150mMTris-HCl, pH 8.0) and mix well, transfer to a 10KD ultrafiltration centrifuge tube, and centrifuge at 12000g for 15min. Add 100μL UAbuffer, centrifuge at 12000g for 10min, repeat twice. Add 100μL 50mM NH4HCO3 buffer, centrifuge at 12000g for 10min, repeat twice. Add an appropriate amount of 20ng / μL Trypsinbuffer (6μg Trypsin in 40μL 50mM NH4HCO3 buffer), shake at 600rpm for 1min, 37℃, 16-18h. Replace the collection tube, centrifuge at 12000g for 10 min, collect the filtrate; desalt the peptides after enzymatic hydrolysis using C18 StageTip and vacuum dry. After drying, re-dissolve the peptides with 0.1% FA and determine the peptide concentration for LC-MS analysis.

[0092] (2) LC-MS / MS

[0093] The peptides were separated using the EasynLC 1200 chromatography system, with solution A (0.1% formic acid aqueous solution) and solution B (a mixed solution containing 0.1% formic acid, 80% acetonitrile and water) as the buffer system. The chromatographic column was balanced with solution A. The sample was injected into the Trap Column (100μm*20mm, 5μm, C18) and then gradient eluted through the analytical column C18 (75μm*150mm) according to Table 2. The separated peptides were analyzed by DDA mode mass spectrometry using a Q-Exactive HF mass spectrometer. The total analysis time was 60min, using the positive ion mode, the parent ion scan range was 350-1800m / z, the resolution of the primary mass spectrometer was set to 120,000@m / z 200, the AGC target value was e6, and the maximum injection time was 50ms. After full scan, the secondary mass spectra of the 20 highest intensity parent ions were selected, the secondary mass spectrometry resolution was set to 15,000@m / z200, the AGC target value was 1e5, the secondary maximum injection time was 50ms, the MS2 activation mode was HCD, the isolation window was set to 1.6m / z, and the normalized collision energy was 28.

[0094] Table 2 EasynLC 1200 HPLC elution gradient

[0095]

[0096] The transcript database of Agkistrodon acutus has been successfully constructed in the early stage of the present invention. After the mass spectrometer data was downloaded, we searched the transcript database. The results showed that a total of 1 protein and 10 peptides were identified. The identified peptide information is shown in Table 3, and the mass spectrum is shown in Figure 7 There are many proteins corresponding to the identified peptides, but the leading razor protein is transcript3439 / f99p0 / 3185. Therefore, the isolated and purified protein is transcript3439 / f99p0 / 3185, which is referred to as 3185 in the following text. 3185 belongs to the serine protease family, with 10 protein peptides, 8 unique peptides, 260 amino acids, a protein molecular weight of 29.1KDa, and an isoelectric point of 8.18.

[0097] The amino acid sequence is as follows:

[0098] MVLIRVLANLLILQLSYAQKSSELIIGGDECDINEHRFLVGLYTSRSRRF

[0099] YCCGTLINPEWVVTAAHCERKNIRIKLGMHSKNTPNEDVQIRVPKEKFFCL

[0100] SSKTYTKWSNDIMLIRLKRPVNNSTHIAPVSLPSNPPSLGSVCRIMGWGTIT

[0101] SPKKTYPDVPHCANILDYEVCREAHPWLPATSRTWCAGILEGGKDTCG

[0102] GDSGGPLICDGQFQGIVSWGWNPCAQQREPGHYTKVIDYNDWIQRNIAGN

[0103] TDATCPP*, SEQ ID NO.1.

[0104] Table 3 Peptide identification table

[0105]

[0106] Example 4

[0107] Activity test of enzyme-cutting chromogenic substrate and hydrolysis of fibrinogen

[0108] (1) Enzyme cleavage chromogenic substrate activity assay

[0109] The activity of purified protein 3185 and defibrase standard on thrombin chromogenic substrate S-2238 was determined by enzyme kinetics. Take S-2238, add PBS to make a 12.5 mg / mL mother solution, divide into portions, and store at -80°C. Dilute purified protein 3185 and defibrase to appropriate concentrations. Incubate S-2238 at a concentration of 0.25 mg / mL at 37°C for 2 minutes, add 90 μL of substrate to a 96-well plate, and immediately add 10 μL of defibrase or purified protein 3185. Detect the absorbance at 405 nm, measure every 30 seconds, and stop the detection after the curve stabilizes.

[0110] (2) Hydrolysis of fibrinogen by purified protein 3185

[0111] Fibrinogen has Aα-chain, Bβ-chain, and γ-chain. Thrombin converts fibrinogen into cross-linked fibrin. It cuts fibrinopeptide A (FPA) from the Aα chain of the fibrinogen molecule to initiate fibrin polymerization. This experiment simulates the in vivo action of thrombin. 200μL of 0.4% coagulable protein solution was incubated at 37°C for 2min, and 200μL of 0.1mg / mL purified protein 3185 was added. The reaction was incubated in a 37°C incubator for 12h and 24h respectively. Each reaction time was repeated three times. The protein was boiled to denature and terminate the reaction. Centrifuged at 3000g for 10min, the supernatant was taken, loading buffer was added, and SDS-PAGE gel electrophoresis was performed using 4% to 20% precast gel. At the same time, 0.4% coagulable protein solution was loaded and the treatment method was the same as the sample. Analyze which chain of fibrinogen the purified protein 3185 acts on.

[0112] The enzyme kinetic curves of the hydrolysis reaction between defibrase and purified protein 3185 and the chromogenic substrate S-2238 are shown in Figure 8 The final concentration of the reaction system was 5 μg / mL, which was the purified protein 3185, and the rest were standard defibrase. Enzyme kinetics is mainly divided into three reaction stages, namely first-order reaction, mixed-order reaction, and zero-order reaction. In the first-order reaction stage, the hydrolysis reaction time of each concentration of defibrase was calculated, and the enzyme concentration and reaction time were linearly fitted on the double logarithmic coordinate paper. The linear equation was y=-0.8591x+2.8162, R 2 =0.9997, and the enzyme concentration of 5 μg / mL purified protein 3185 is 5.16 U / mL, that is, the specific activity of purified protein 3185 is 1032 U / mg.

[0113] SDS-PAGE electrophoresis of hydrolyzed fibrinogen from purified protein 3185 ( Fig. 9 ) In the lane, fibrinogen showed three clear chains of α, β, and γ. After the purified protein 3185 reacted with fibrinogen, only the Aα chain was degraded, and the degree of degradation increased with time. The hydrolysis of fibrinogen was consistent with that of defibrase.

[0114] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0115] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A use of a snake venom thrombin protein in the preparation of an anticoagulant drug, characterized in that: The amino acid sequence of the snake venom thrombin-like protein is as follows: MVLIRVLANLLILQLSYAQKSSELIIGGDECDINEHRFLVGLYTSRSRRFYCCGTLINPEWVVTAAHCERKNIRIKLGMHSKNTPNEDVQIRVPKEKFFCLSSKTYTKWSNDIMLIRLKRPVNNSTHIAPVS LPSNPPSLGSVCRIMGWGTITSPKKTYPDVPHCANINILDYEVCREAHPWLPATSRTWCAGILEGGKDTCGGDSGGPLICDGQFQGIVSWGWNPCAQQREPGHYTKVIDYNDWIQRNIAGNTDATCPP*, SEQ ID NO.

1.

2. The use according to claim 1, characterized in that Includes the following peptides: EAHPWLPATSR, SEQ ID NO. 2; IMGWGTITSPK, SEQ ID NO. 3; KTYPDVPHCANILDYEVCR,SEQ ID NO.4; NTPNEDVQIRVPK, SEQ ID NO.5; TWCAGILEGGK, SEQ ID NO. 6; TYPDVPHCANINILDYEVCR,SEQ ID NO.7; VIDYNDWIQR, SEQ ID NO. 8; WSNDIMLIR; FFCLSSK; FLVGLYTSR.

3. The use according to claim 1, characterized in that The preparation method of the snake venom thrombin-like protein is as follows: The snake venom freeze-dried powder was separated and purified by SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, SUPERDEX75INCREASE 10 / 300GL gel chromatography column, C18 SP-100-5-ODS-P liquid chromatography column and C18 SP-100-5-ODS-P liquid chromatography column in sequence.

4. The use according to claim 3, characterized in that The preparation method of the snake venom thrombin-like protein comprises the following steps: (1) The lyophilized snake venom powder was isocratically eluted through a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, with the eluent being a PBS solution, and the fraction with the highest thrombin activity was collected; (2) isocratically eluting the fraction obtained in step (1) through a SUPERDEX 75INCREASE 10 / 300GL gel chromatography column, using a PBS solution as the eluent, and collecting the fraction with the highest thrombin activity; (3) gradient eluting the components obtained in step (2) through a C18 SP-100-5-ODS-P liquid chromatography column with a mobile phase system of water and acetonitrile, and collecting the components with the highest thrombin activity; (4) The components obtained in step (3) are subjected to gradient elution through a C18 SP-100-5-ODS-P liquid chromatography column with a mobile phase system of water and acetonitrile, and the components with the highest thrombin activity are collected.

5. The use according to claim 4, characterized in that The elution flow rate of steps (1) and (2) is 0.8 mL / min.

6. The use according to claim 4, characterized in that The elution flow rate of steps (3) and (4) is 2 mL / min.

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