Hemocoagulase extracted from the venom of the spearhead viper, its preparation method, and its uses.

Abstract

This invention provides a hemocoagulase extracted from the venom of the Bothrops atrox. The invention also provides a method for preparing the hemocoagulase, comprising the following steps: pretreating the Bothrops atrox venom, followed by sequentially passing it through diethylaminoethyl cross-linked dextran gel A-25 ion exchange chromatography; benzalkonium agarose gel 4FF (HS) affinity chromatography; and cross-linked dextran gel G-75 gel filtration chromatography to obtain the Bothrops atrox venom hemocoagulase. This invention also provides the application of the Bothrops atrox venom hemocoagulase in the preparation of drugs for treating hemorrhagic diseases. Compared with existing technologies, the preparation process of this invention has fewer steps, a shorter cycle, and reduces the risk of environmental exposure during the process; it has a high product yield, strong impurity removal capability, and can obtain Bothrops atrox hemocoagulase with a yield of over 35% and a purity of over 98%; the process has high stability, good product quality consistency, and is beneficial to clinical drug safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for extracting hemocoagulase from the venom of the lancehead viper. This invention also relates to a method for extracting hemocoagulase from the venom of the lancehead viper and the uses of the hemocoagulase. Background Technology

[0002] Coagulation is a complex physiological process, which can be roughly divided into three stages: the first stage is the activation of coagulation factor X (FX); the second stage is the activation of prothrombin; and the third stage is the conversion of fibrinogen into fibrin.

[0003] Extracting hemostatic agents from snake venom has a long history. Currently, Reptilase, a hemostatic drug produced in Switzerland, is one of the more successful in clinical applications. Its main component is a snake venom thrombin-like enzyme (SVTLE) extracted from the venom of the Brazilian snake (Bothrops jrarace, Lachesisatrox). This enzyme belongs to the serine protease family of trypsinase and possesses arginine esterase and amidase activities. In vitro, this enzyme hydrolyzes fibrinogen into fibrin without the participation of other coagulation factors. In vivo, it does not activate coagulation factor XIII, and the resulting fibrin clot is unstable and easily dissolved by the fibrinolytic system, thus not affecting platelet activation and release and posing no risk of thrombosis. Because this thrombin-like enzyme is structurally and functionally similar to human thrombin, it is called "thrombin-like enzyme." To date, thrombin-like enzymes have been discovered in more than 30 snake venoms, and more than 20 of them have been successfully isolated and purified. Among them, the complete or partial amino acid sequences of more than 10 thrombins have been elucidated (Zheng Ying, Shen Juren, Zhang Fuqiang, et al. Determination of N-terminal sequence of thrombin from pit viper and analysis of its hemostatic activity [J]. Journal of China Medical University).

[0004] Since most of the thrombin-like substances extracted from natural snake venom are acidic proteins, they are often separated using anion exchange columns, and purified using methods such as gel filtration, affinity chromatography, and reversed-phase liquid chromatography.

[0005] In the reported literature, the methods for extracting and purifying snake venom thrombin include:

[0006] 1) After pretreatment of snake venom, separation is performed using diethylaminoethyl-dextran gel A50 (DEAE-Sephadex A50) anion exchange chromatography and Sephadex G 100 molecular sieve chromatography. This pretreatment process is cumbersome, resulting in significant loss of enzyme active components, affecting the specific activity and yield of the product. Furthermore, the use of organic solvents such as phenol and its derivatives, and methanol in the pretreatment process affects subsequent purification, making it unsuitable for large-scale production.

[0007] 2) After pretreatment of snake venom, heparin-CNBr-Sepharose 4B affinity chromatography is performed. Although this pretreatment method is slightly simpler than the previous method, it still uses phenol derivatives in the pretreatment, and the process activity yield is low, only 7.4%, making it unsuitable for large-scale production.

[0008] Chinese patent application No. 200610044594.1 discloses a method for extracting batroxobin, a single component, from the venom of the pit viper. It discloses a method for purifying the pit viper venom by dissolving, overnight incubation, centrifugation, Benzamidine Sepharose 6B affinity chromatography, Sephadex G-25 chromatography, dialysis, cation exchange chromatography (SP Sepharose, S Sepharose, or CM Sepharose), and molecular sieve chromatography (Sephacry1S200, Sephacry1S100, or Superdex-75) to obtain the single component batroxobin. However, the chromatographic steps in this patent are cumbersome, and the cation exchange column uses either staged gradient elution or linear gradient elution, which is not conducive to the removal of pyrogens and impurities in large-scale production.

[0009] Chinese patent application CN200710099163.X discloses a hemagglutinin from the venom of the white-browed viper (Gnaphalium affine) and its extraction method and application. It discloses that this hemagglutinin is a protein with coagulation activity collected by sequentially subjecting the venom of the white-browed viper (Gnaphalium affine) to DEAE-Sephadex A-50 ion exchange chromatography, Sephadex G-15 gel filtration chromatography, DEAE-Sephadex A-50 ion exchange column chromatography, and Sephadex G-75 gel filtration chromatography. However, this patent application employs a four-step chromatographic method, and the chromatographic processes are mostly linear gradient elution or staged gradient elution, making the process complex.

[0010] Chinese patent application No. 200510085173.9 discloses a method for preparing and using hemocoagulase from the pit viper. It discloses a process including dissolving snake venom, low-temperature centrifugation, dialysis, DEAE-Sepharose FF chromatography, dialysis again, DEAE-Sepharose FF chromatography, and Sephadex G25 chromatography. Although the disclosed method involves three steps in the hemocoagulase purification chromatography, it employs gradient elution, resulting in low yields or ineffective pyrogen removal during the purification process while maintaining purity.

[0011] In summary, the extraction methods for snake venom hemagglutinin disclosed in existing literature or patent applications are difficult to achieve the ideal separation and preparation requirements, and are not suitable for large-scale production. Summary of the Invention

[0012] Therefore, the purpose of this invention is to provide a method for extracting hemocoagulase from the venom of the pit viper. This invention also provides the uses of the hemocoagulase from this invention. The method for extracting hemocoagulase provided by this invention has fewer steps and a shorter cycle, thereby reducing the risk of environmental exposure during the process. Furthermore, the method provided by this invention has strong impurity removal capabilities and a high yield, enabling the acquisition of pit viper hemocoagulase with a yield of over 35% and a purity of over 98%.

[0013] The objective of this invention is achieved by a method comprising the following steps.

[0014] On one hand, the present invention provides a hemagglutinin derived from the venom of the lancehead viper, which has the following characteristics:

[0015] 1) The molecular weight of hemocoagulase is 31731 Da, and after sugar removal, the molecular weight is 25575 Da. The N-glycan content is 19.4%, and the three N-glycosylation sites are N... 98 N 146 With N 225 ;

[0016] 2) The isoelectric point is 5-6;

[0017] 3) The N-terminal sequence of hemocoagulase is: VIGGDECDINEHPFL;

[0018] 4) Hemocoagulase contains 6 pairs of disulfide bonds and has no free sulfhydryl groups;

[0019] 5) The hemocoagulase protein sequence contains 232 amino acids.

[0020] Preferably, the amino acid sequence of the hemocoagulase is shown in SEQ ID NO:1:

[0021] VIGGDECDINEHPFLAFMYYSPQYFCGMTLINQEWVLTAAHCDKTYMRIYLGIHTRSVANDDEVIRYPKEKFICPNKKKNVITDKDIMLIRLNRPVKNSTHIAPISLPSNPPSVGS VCRIMGWGAITTSEDTYPDVPHCANINLFNNTVCREAYNGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCPP.

[0022] On the other hand, the present invention provides a method for extracting hemagglutinin from the venom of the spearhead viper, the method comprising the following steps:

[0023] After pretreatment, the venom of the pit viper was subjected to sequential ion exchange chromatography on diethylaminoethyl cross-linked dextran gel (DEAESephadex A-25); affinity chromatography on Benzamidine Sepharose 4FF(HS); and gel filtration chromatography on cross-linked dextran gel (Sephadex G-75) to obtain the stock solution of pit viper venom hemocoagulase.

[0024] Preferably, the method includes the following steps:

[0025] 1) Dissolve the venom of the spearhead viper in a 0.01M-0.05M Tris-HCl buffer solution at pH 7.0-7.5 to obtain a solution with a concentration of 50-200 mg / ml, preferably 80-150 mg / ml, and more preferably 100 mg / ml, and then centrifuge to collect the supernatant.

[0026] Preferably, the Tris-HCl buffer solution has a pH of 7.4 and a concentration of 0.02 M;

[0027] Preferably, the centrifugation conditions are: 4°C, 3000rpm-5000rpm, centrifugation time of 5-20min, preferably 10min;

[0028] 2) Perform DEAE Sephadex A-25 ion exchange chromatography on the supernatant obtained in step 1). Use Tris-HCl buffer containing 0.2M-0.4M NaCl, pH 7.0-7.5, and concentration 0.01M-0.05M for linear elution. Collect the eluent based on the absorption peak at 280nm wavelength to obtain the eluent containing coagulation active components.

[0029] Preferably, the Tris-HCl buffer solution has a pH of 7.4 and a concentration of 0.02 M;

[0030] Preferably, the concentration of NaCl in the Tris-HCl buffer solution is 0.3M;

[0031] 3) The eluent containing the coagulation-active component obtained in step 2) is dialyzed with a Tris-HCl buffer containing 0.5M-1.5M NaCl, pH 7.0-7.5, and concentration 0.01-0.06M, followed by Benzamidine Sepharose 4FF (HS) affinity chromatography, eluting with a Tris-HCl buffer containing 0.05-0.3M arginine, 0.5M-1.5M NaCl, pH 7.0-7.5, and concentration 0.01-0.06M, and the target component is collected; preferably, the Tris-HCl buffer has a pH of 7.0 and a concentration of 0.05M.

[0032] Preferably, the concentration of arginine in the Tris-HCl buffer solution is 0.1M;

[0033] Preferably, the concentration of NaCl in the Tris-HCl buffer solution is 1.0 M;

[0034] 4) The target component collected in step 3) was subjected to Sephadex G-75 gel filtration chromatography, and eluted with phosphate buffer at pH 7.0-7.5 and a concentration of 0.05M-0.3M. The eluent was collected to obtain the stock solution of lancehead viper venom hemagglutinin.

[0035] Preferably, the phosphate buffer solution has a pH of 7.4 and a concentration of 0.1M;

[0036] Preferably, in step 2), before performing DEAE Sephadex A-25 ion exchange chromatography, three column volumes are equilibrated with Tris-HCl buffer at pH 7.0-7.5 and a concentration of 0.01M-0.05M; preferably, the column flow rate is 0.8-3 ml / min, more preferably 2 ml / min; preferably, the Tris-HCl buffer has a pH of 7.4 and a concentration of 0.02M.

[0037] Preferably, in step 2), the eluent containing the coagulation active component is detected using a quality control plasma coagulation analyzer activity detection method (as described in patent CN103305591A) and SDS-PAGE (Chinese Pharmacopoeia 2015 edition, Part IV, General Chapter 0541, protein molecular weight 36000±5000 Da) electrophoresis to determine the target component.

[0038] Preferably, in step 3), before performing Benzamidine Sepharose 4FF (HS) affinity chromatography, dialyzing is performed using a Tris-HCl buffer containing 0.5M-1.5M NaCl, with a pH of 7.0-7.5 and a concentration of 0.01-0.06M. The eluent containing the coagulation-active component is aseptically dialyzed through an ultrafiltration membrane 2-3 times. More preferably, the pH of the Tris-HCl buffer is 7.0 and the concentration is 0.05M. Even more preferably, the concentration of NaCl in the Tris-HCl buffer is 1.0M. Preferably, the ultrafiltration membrane is an ultrafiltration membrane package. More preferably, the molecular weight cutoff of the ultrafiltration membrane is 10K. Even more preferably, the ultrafiltration membrane is made of polyethersulfone.

[0039] Preferably, in step 3), before using the Benzamidine Sepharose 4FF(HS) affinity chromatography column, three column volumes are equilibrated with a Tris-HCl buffer containing 0.5M-1.5M NaCl, with a pH of 7.0-7.5 and a concentration of 0.01-0.06M; preferably, the column flow rate is 1.0ml-3ml / min, more preferably 2ml / min; preferably, the pH of the Tris-HCl buffer is 7.0 and the concentration is 0.05M; preferably, the concentration of NaCl in the Tris-HCl buffer is 1.0M.

[0040] Preferably, in step 4), before performing Sephadex G-75 gel filtration chromatography, three column volumes are equilibrated with a phosphate buffer solution of pH 7.0-7.5 and a concentration of 0.05M-0.3M; preferably, the column flow rate is 0.8-3 ml / min, more preferably 2 ml / min; preferably, the phosphate buffer solution has a pH of 7.4 and a concentration of 0.1M.

[0041] Preferably, in step 4), after chromatography, the eluent is concentrated using a dialysis bag with a molecular weight cutoff of 10k; preferably, polyethylene glycol is used as the reverse osmosis agent.

[0042] Preferably, the obtained lancehead viper venom hemocoagulase stock solution is filtered, dispensed, and freeze-dried to obtain lancehead viper venom hemocoagulase freeze-dried powder.

[0043] Preferably, the lyophilized powder of *Viper lanceolata* venom hemocoagulase is prepared together with excipients and a protective agent to obtain an injectable *Viper lanceolata* venom hemocoagulase preparation.

[0044] In another aspect, the present invention provides the application of the lancehead viper venom hemocoagulase described in the present invention and the lancehead viper venom hemocoagulase prepared according to the above method in the preparation of a drug for treating hemorrhagic diseases.

[0045] Preferably, the bleeding includes clinical bleeding, surgical bleeding, and various acute internal and surgical bleeding.

[0046] On the other hand, the present invention provides a pharmaceutical composition for treating bleeding, the pharmaceutical composition comprising the lancehead viper venom hemocoagulase described in the present invention and / or the lancehead viper venom hemocoagulase prepared according to the above method, and a pharmaceutically acceptable carrier.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] 1) The preparation process of this invention has fewer steps and a shorter cycle, reducing the risk of environmental exposure during the process;

[0049] 2) The product yield is high, and the process has a strong ability to remove impurities, enabling the production of lancehead viper hemagglutinin with a yield of over 35% and a purity of over 98%.

[0050] 3) The product has a high specific activity, and each unit of sample contains less protein, thus avoiding immunogenic reactions;

[0051] 4) The process has high stability and good product quality consistency, which is conducive to the safety of clinical drug use. Attached Figure Description

[0052] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0053] Figure 1 Mass spectrum of hemagglutinin obtained by MALDI-TOF mass spectrometry;

[0054] Figure 2 Mass spectra of hemocoagulase after deglycated and after deglycated and NEM alkylation, determined by ESI-Q-TOF mass spectrometry.

[0055] Figure 3 The RP-HPLC purity of the lancehead viper hemagglutinin extracted using the method of the present invention;

[0056] Figure 4 SDS-PAGE purity of the lancehead viper hemagglutinin extracted using the method of the present invention;

[0057] Figure 5 SDS-PAGE purity of hemagglutinin extracted from Changbai Mountain white-browed viper using the method of the present invention;

[0058] Figure 6 SDS-PAGE purity of hemagglutinin extracted from lancelet viper using existing methods;

[0059] Figure 7 Fibrin formed by the hemagglutinin of the venom of the spearhead viper and fibrin filaments after digestion with fibrinolytic enzyme;

[0060] Figure 8 Fibrin formed from human thrombin and fibrin filaments after being digested with plasmin. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0062] According to the method described in this invention, hemagglutinin with a purity of over 98% can be prepared. The molecular weight of the hemagglutinin was then determined to be 31731 Da using MALDI-TOF mass spectrometry (Bruker microTOF-Q II mass spectrometer), and the molecular weight after sugar removal was determined to be 25575 Da using ESI-Q-TOF mass spectrometry (Bruker microTOF-Q II mass spectrometer). The N-sugar content was 19.4%, it contained 6 pairs of disulfide bonds, and had no free sulfhydryl groups; it possessed 3 N-glycosylation sites, namely N... 98 N 146 With N 225 The isoelectric point was determined to be 5-6 by solid-phase pH gradient (IPG) isoelectric focusing (Bio-Rad manual); the first 15 amino acids of the N-terminus of hemagglutinin were determined by Edman sequencer to be: VIGGDECDINEHPFL.

[0063] The protein sequence contains 232 amino acids, as shown in SEQ ID NO:1:

[0064] SEQ ID NO:1:

[0065] VIGGDECDINEHPFLAFMYYSPQYFCGMTLINQEWVLTAAHCDKTYMRIYLGIHTRSVANDDEVIRYPKEKFICPNKKKNVITDKDIMLIRLNRPVKNSTHIAPISLPSNPPSVGS VCRIMGWGAITTSEDTYPDVPHCANINLFNNTVCREAYNGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCPP.

[0066] Furthermore, see details. Figure 1 and Figure 2 ,in Figure 1 Mass spectrum of hemagglutinin obtained by MALDI-TOF mass spectrometry; Figure 2 Mass spectra of hemocoagulase after deglycination and after deglycination and NEM alkylation, determined by ESI-Q-TOF mass spectrometry.

[0067] The present invention also uses pharmacodynamic tests to demonstrate that the hemocoagulant of the present invention has hemostatic effects.

[0068] Example 1: Extraction of hemagglutinin from the venom of the spearhead viper.

[0069] Extraction steps

[0070] 1. Snake venom pretreatment

[0071] Accurately weigh 80g of lancehead viper venom (purchased from Liaoning Yuanda Nuokang Biopharmaceutical Co., Ltd.), dissolve it in pH 7.4, 0.02M Tris-HCl buffer to obtain a 100mg / ml solution, centrifuge at 4000rpm at 4℃ for 10min, and collect the supernatant.

[0072] 2. DEAE Sephadex A-25 ion exchange chromatography (column I, purchased from GE).

[0073] Before loading the chromatography column (4.5×50cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with pH 7.4, 0.02M Tris-HCl buffer at a flow rate of 2 ml / min. Check the bacterial endotoxin level in the backfill solution; if the endotoxin level does not exceed 0.25 EU / ml, it is ready for sample loading.

[0074] Take the supernatant from step 1 and load it onto the sample. Elute with 2 column volumes of pH 7.4, 0.02M Tris-HCl buffer. Then, perform linear elution with the above Tris-HCl buffer of pH 7.4, 0.4M NaCl at a flow rate of about 3 ml / min. Collect the protein based on the absorption peak at 280 nm.

[0075] 3. Benzamidine Sepharose 4FF (HS) affinity column chromatography (column II, purchased from GE)

[0076] The collected eluted fractions were identified by activity determination using a quality control plasma coagulation analyzer (patent CN103305591A) and SDS-PAGE (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0541) electrophoresis. Target fractions with thrombin activity above 100 units / ml and protein molecular weight of 36000±5000 Da were collected. The fractions were aseptically dialyzed three times using an ultrafiltration membrane (Sartorius, polyethersulfone, molecular weight cutoff 10K) with 0.05M Tris-HCl buffer (pH 7.0, 1.0M NaCl). Before loading the chromatography column (1.6×30cm, Shanghai Jinhua glass chromatography column), the column was equilibrated with the above buffer for three column volumes. The collected target fractions were then loaded at a flow rate of 2 ml / min. After eluting with the above buffer for two column volumes, elution was performed with 0.05M Tris-HCl eluent (pH 7.0, containing 0.1M arginine and 1.0M NaCl) at a flow rate of 2 ml / min. Protein peaks were collected based on the absorbance at 280 nm wavelength, and the target active components were determined by activity assay using a quality control plasma coagulation analyzer and SDS-PAGE electrophoresis.

[0077] 4. Sephadex G-75 column chromatography (column III, purchased from GE)

[0078] Before loading the chromatography column (4.5×100cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with 0.1M phosphate buffer (pH 7.4) at a flow rate of 2 ml / min. Check that the bacterial endotoxin level in the bottom solution does not exceed 0.25 EU / ml before loading. Take the affinity chromatography active fraction and concentrate it using a dialysis bag with a molecular weight cutoff of 10k. Use polyethylene glycol as the reverse osmosis solvent. After loading, elute with the above buffer at a rate of 2 ml / min. Detect the eluent with a UV detector and collect the protein peak fraction containing hemocoagulase.

[0079] Results Analysis

[0080] 1. Purity testing

[0081] Detection was performed using an electrophoresis system and reversed-phase high-performance liquid chromatography (Shimadzu HPLC 20A, C4 column). Denaturing non-reducing electrophoresis using SDS-PAGE showed the target protein component as a single band (e.g., ...). Figure 4 As shown), RP-HPLC detection showed a single symmetrical peak (e.g. Figure 3 As shown in the figure, the hemagglutinin content is 99.8% according to the area normalization method.

[0082] 2. Target protein activity was detected by a quality control plasma coagulation analyzer. The total activity of hemocoagulase in the solution after dissolving 80g of snake venom was 5.76 million units. After chromatographic purification, the collected hemocoagulase activity was 2.2 million units, with a yield of 38.2%.

[0083] 3. Detection of protein content of target components

[0084] The hemocoagulase was tested using the Folin-Ciocalteu method (Chinese Pharmacopoeia, Part IV, General Chapter 0731, Protein Content Determination Method), and the protein content was 475 mg, with a specific activity of 4632 units / mg.

[0085] 4. Preparation of stock solution

[0086] The collected protein components with qualified purity were dialyzed three times with water for injection, sterilized by filtration through a 0.22μm microporous membrane, aseptically dispensed, and freeze-dried to obtain lyophilized lancehead viper hemagglutinin powder.

[0087] 5. Structural characteristics of hemagglutinin from the lancehead viper

[0088] A. MALDI-TOF mass spectrometry determined the molecular weight of hemagglutinin to be 31731 Da, while ESI-Q-TOF mass spectrometry determined the molecular weight after sugar removal to be 25575 Da. The N-glycan content was 19.4%, containing 6 disulfide bonds and no free sulfhydryl groups; it also possessed 3 N-glycosylation sites, namely N... 98 N 146 With N 225 ;

[0089] B. The isoelectric point measured by solid phase pH gradient (IPG) isoelectric focusing method (Bio-Rad manual) is 5-6;

[0090] The C.Edman sequencer determined the first 15 amino acids of the N-terminus of hemagglutinin to be: VIGGDECDINEHPFL;

[0091] D. The hemocoagulase protein sequence contains 232 amino acids.

[0092] VIGGDECDINEHPFLAFMYYSPQYFCGMTLINQEWVLTAAHCDKTYMRIYLGIHTRSVANDDEVIRYPKEKFICPNKKKNVITDKDIMLIRLNRPVKNSTHIAPISLPSNPPSVGS VCRIMGWGAITTSEDTYPDVPHCANINLFNNTVCREAYNGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCPP.

[0093] Example 2: Extraction of hemagglutinin from the venom of the spearhead viper.

[0094] Extraction steps

[0095] 1. Snake venom pretreatment

[0096] Accurately weigh 80g of lancehead viper venom and dissolve it in pH 7.0, 0.04M Tris-HCl buffer to obtain a solution with a concentration of 200mg / ml. Centrifuge at 3000rpm at 4℃ for 20min and collect the supernatant.

[0097] 2. DEAE Sephadex A-25 ion exchange chromatography (column I)

[0098] Before loading the chromatography column (4.5×50cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with pH 7.0, 0.04M Tris-HCl buffer at a flow rate of 2 ml / min. Check the bacterial endotoxin level in the backfill solution; if the endotoxin level does not exceed 0.25 EU / ml, it is ready for sample loading.

[0099] Take the supernatant from step 1 and load it onto the sample. Elute for 2 column volumes with 0.01M Tris-HCl buffer at pH 7.0. Then, perform linear elution with the above Tris-HCl buffer at pH 7.0 and 0.2M NaCl at a flow rate of 3 ml / min. Collect the protein based on the absorption peak at 280 nm.

[0100] 3. Benzamidine Sepharose 4FF(HS) affinity column chromatography (column II)

[0101] The collected eluted fractions were identified by activity determination using a quality control plasma coagulation analyzer (patent CN103305591A) and SDS-PAGE (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0541) electrophoresis. Target fractions with thrombin activity above 100 units / ml and protein molecular weight of 36000±5000 Da were collected. The fractions were aseptically dialyzed three times using an ultrafiltration membrane (Sartorius, polyethersulfone, molecular weight cutoff 10K) with 0.05M Tris-HCl buffer containing pH 7.0 and 1.5M NaCl. Before loading the chromatography column (1.6×30cm, Shanghai Jinhua glass chromatography column), the column volume was equilibrated with the above buffer for three column volumes, and the collected target fractions were loaded at a flow rate of 2 ml / min. After eluting for two column volumes with the above buffer, elution was performed with 0.05M Tris-HCl elution buffer (pH 7.0) containing 0.05M arginine and 1.5M NaCl at a flow rate of 2 ml / min. Protein peaks were collected based on absorbance at 280 nm, and the target active component was identified by activity assay using a quality control plasma coagulation analyzer and SDS-PAGE electrophoresis.

[0102] 4. Sephadex G-75 column chromatography (column III)

[0103] Before loading the chromatography column (4.5×100cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with 0.05M phosphate buffer (pH 7.4) at a flow rate of 2 ml / min. Check that the bacterial endotoxin level in the bottom solution does not exceed 0.25 EU / ml before loading. Take the affinity chromatography active fraction and concentrate it using a dialysis bag with a molecular weight cutoff of 10k, with polyethylene glycol as the reverse osmosis solvent. Elute with the above buffer at a rate of 2 ml / min. Detect the eluent with a UV detector and collect the protein peak fraction containing hemocoagulase.

[0104] Results Analysis

[0105] 1. Purity testing

[0106] Electrophoresis and reversed-phase high-performance liquid chromatography (RP-HPLC) were used for detection. SDS-PAGE denaturing non-reducing electrophoresis showed a single band for the target protein component, while RP-HPLC showed a single symmetrical peak. The hemagglutinin content was calculated to be 98.2% using the area normalization method.

[0107] 2. Target protein activity detection

[0108] The total activity of hemocoagulase in the solution after dissolving 80g of snake venom was 5.82 million units, as determined by the quality control plasma coagulation analyzer method. After chromatographic purification, the activity of the collected hemocoagulase was 2.05 million units, with a yield of 35.2%.

[0109] 3. Detection of protein content of target components

[0110] The hemocoagulase was detected by the Folin-Ciocalteu method (Chinese Pharmacopoeia, Part IV, General Chapter 0731, Protein Content Determination Method), and the protein content was 502 mg, with a specific activity of 4084 units / mg.

[0111] Example 3: Study on the coagulation function of hemocoagulase

[0112] This study investigated the effects of lancehead viper hemocoagulase on coagulation function and fibrin formation in patients with hemorrhagic diseases in vitro.

[0113] Experiment 1

[0114] 1. Experimental Objective

[0115] This study investigated the effects of lancehead viper hemocoagulase on activated partial thromboplastin time (APTT), prothrombin time (PT), factor X activation (FX), and thrombin formation in patients with hemorrhagic diseases.

[0116] 2. Experimental subjects and methods

[0117] Specimen preparation and reagents: In accordance with informed consent, 3.8% sodium citrate anticoagulated plasma was collected from 20 healthy individuals aged 20-45 years, with an equal number of males and females. The participants included 25 patients with hemophilia A, 7 patients with vitamin K-dependent clotting factor deficiency, 3 patients with von Willebrand's disease (VWD), 3 patients with FX deficiency, and 1 patient each with hemophilia B, FX V, FX VII, and fibrinogen deficiency. The plasma was aliquoted and stored at -20°C for later use.

[0118] Test drug: Viper hemagglutinin, prepared according to the method described in Example 1 of the present invention;

[0119] Human plasma coagulation factor X (FX), manufactured by Calbiochem;

[0120] Color-developing substrates S-2337 and S-2238, manufactured by Sigma;

[0121] Activated FXa (FXa) standard and human thrombin standard, manufactured by Hyphen BioMed.

[0122] Instruments: STAGO COMPACT fully automated blood coagulation analyzer (Stago, France); multi-wavelength microplate reader (Cermolfess Technology); KDC-40 low-speed centrifuge (Beijing Southeast Yicheng Laboratory Equipment Co., Ltd.)

[0123] 3. Experimental Methods

[0124] 3.1 Detection of Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT)

[0125] A certain concentration of lancehead viper hemagglutinin was added to the plasma of normal individuals and patients with hemorrhagic diseases, respectively. After incubation at 37°C for 3 minutes, APTT and PT were measured on a fully automated hemagglutination analyzer and compared with a blank control group (with an equal volume of physiological saline added). (APTT reference range: 28s-40s, PT reference range: 11s-14.5s.)

[0126] 3.2 Chromogenic substrate method for detecting plasma FX activation and thrombin generation

[0127] S-2337 and S-2238 are specific chromogenic substrates for FXa and thrombin, respectively. After hydrolysis, they release the chromogenic group p-nitroaniline. The amount of p-nitroaniline released is proportional to the activity of FXa and thrombin. An absorption peak was detected at 405 nm.

[0128] Take 100 μl of plasma or FX (8 μg / ml) and add 100 μl of lancehead viper hemocoagulase solution [lancehead viper hemocoagulase 0.02 U / ml, Tris-HCl 100 mmol / L (pH 7.5), CaCl2 6 mmol / L, anisole hydrochloride 2 mmol / L], incubate at 37℃ for 5 min, then add 100 μl to a 96-well microplate, and add 100 μl of chromogenic substrate buffer [S-2337 0.625 mg / ml, Tris-HCl 0.5 mmol / L (pH 8.3), EDTA 25 mmol / L, NaCl 375 mmol / L], and measure the absorbance at 405 nm for 30 min.

[0129] Take 100 μl of plasma, add 1 μl of FXA (0.1 U / ml) solution, incubate at 37℃ for 5 min, then spot 10 μl onto a 96-well microplate, add 96 μl of chromogenic substrate buffer (S-2238 2 mmol / L, EDTA 5 mmol / L, benzidine hydrochloride 50 μmol / L, CaCl2 3 mmol / L), and measure the absorbance at 405 nm for 30 min.

[0130] 4. Experimental Results:

[0131] The lancehead viper thrombin can shorten the acute pulse time (APTT) of normal human plasma. At a concentration of 0.05 U / ml, it can shorten the APTT of normal human plasma by about 10 seconds. The lancehead viper thrombin can also shorten the APTT of hemophiliac plasma in a dose-dependent manner, reaching normal levels at a concentration of 0.05 U / ml (as shown in Table 1). The lancehead viper thrombin has no significant effect on prothrombin time (PT) in normal individuals and patients with coagulation disorders (as shown in Table 2).

[0132] Table 1. Effects of lancehead viper hemocoagulase on APTT in healthy individuals and hemophiliac patients.

[0133]

[0134] *P<0.05, ***P<0.001 vs hemophilia patients (n=25); ###P<0.001 vs normal individuals (n=6)

[0135] Table 2. Effects of lancehead viper hemocoagulase on PT in normal individuals and patients with coagulation disorders.

[0136]

[0137] The thrombin from the lancehead viper has no significant effect on plasma FX activation and thrombin generation in normal individuals and patients with coagulation disorders.

[0138] 5. Conclusion

[0139] The thrombin from the lancehead viper can significantly shorten the plasma APTT time in normal individuals and those with coagulation disorders, but has no significant effect on PT, FX activation, or thrombin generation.

[0140] Experiment 2

[0141] 1. Experimental Objective

[0142] The effect of hemocoagulase from the lancet viper on fibrin formation was investigated using scanning electron microscopy.

[0143] 2. Experimental Materials and Methods

[0144] Based on the principle of informed consent, 3.8% sodium citrate anticoagulated plasma was collected from 20 healthy individuals aged 20-45 years, with an equal number of men and women.

[0145] Test drugs: Viper lancet hemocoagulase, prepared according to the method described in Example 1 of this invention; human thrombin, purchased from Sigma;

[0146] Scanning electron microscope: CamScan 3400;

[0147] Scanning electron microscopy (SEM) procedure: 500 μl of normal human plasma was placed into four 1.5 ml centrifuge tubes and labeled. 5 μl of 5 U / ml viper thrombin was added to tubes 1 and 2, and 5 μl of 500 U / ml human thrombin was added to tubes 3 and 4. Each tube was incubated at 37°C for 30 min. Then, 5 μl of 5 mg / ml plasminogen activator (rt-PA) was added to tubes 2 and 4, and the mixture was gently stirred and incubated for 15 min. Fibrin clots were gently removed from each tube, washed three times with physiological saline, fixed with 2.5% glutaraldehyde solution, and sent for SEM examination (see figures below). Figure 7 and Figure 8 (As shown). The width of the fibrin filaments was statistically analyzed by computer.

[0148] 3. Experimental Results

[0149] Electron microscopy results showed that the thrombin from the lancehead viper can promote the formation of relatively fine fibrin filaments (153.5±12.4 μm). These fibrin filaments, after being digested by plasmin, became blurred and swollen; they were more easily digested than those formed by thrombin. This indicates that the lancehead viper thrombin, unlike human thrombin, promotes fibrin formation at the bleeding site that is unstable and easily degraded by plasmin, thus reducing the likelihood of thrombotic complications.

[0150] 4. Conclusion

[0151] The thrombin from the spearhead viper can promote the formation of easily digestible fibrinogen. Compared to thrombin, it is less likely to form blood clots.

[0152] Comparative Example 1: Extraction of hemagglutinin from the venom of the white-browed viper (Viper venom) of Changbai Mountain

[0153] Extraction steps

[0154] 1. Snake venom pretreatment

[0155] Accurately weigh 80g of white-browed viper venom and dissolve it in a 0.02M Tris-HCl buffer solution at pH 7.4 to obtain a 100mg / ml solution. Centrifuge at 4000rpm at 4℃ for 10min and collect the supernatant.

[0156] 2. DEAE Sephadex A-25 ion exchange chromatography (column I)

[0157] Before loading the chromatography column (4.5×50cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with pH 7.4, 0.02M Tris-HCl buffer at a flow rate of 2 ml / min. Check the bacterial endotoxin level in the backfill solution; if the endotoxin level does not exceed 0.25 EU / ml, it is ready for sample loading.

[0158] Take the supernatant from step 1 and load it onto the sample. Elute with 0.02M Tris-HCl buffer at pH 7.4 for 2 column volumes, then elute linearly with the above Tris-HCl buffer at pH 7.4 and 0.4M NaCl at a flow rate of 3 ml / min. Collect the sample based on the absorption peak at 280 nm.

[0159] 3. Benzamidine Sepharose 4FF(HS) affinity column chromatography (column II)

[0160] The collected eluted fractions were identified by activity determination using a quality control plasma coagulation analyzer (patent CN103305591A) and SDS-PAGE (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0541) electrophoresis. Target fractions with thrombin activity above 100 units / ml and protein molecular weights of 36000-43000 Da were collected. The fractions were aseptically dialyzed three times using an ultrafiltration membrane (Sartorius, polyethersulfone, molecular weight cutoff 10K) with 0.05M Tris-HCl buffer containing pH 7.0 and 1.0M NaCl. Before loading the chromatography column (1.6×30cm, Shanghai Jinhua glass chromatography column), the column volume was equilibrated with the above buffer for three column volumes, and the collected target fractions were loaded at a flow rate of 2 ml / min. After eluting for two column volumes with the above buffer, elution was performed with 0.05 M Tris-HCl elution buffer (pH 7.0) containing 0.1 M arginine and 1.0 M NaCl at a flow rate of 2 ml / min. Protein peaks were collected based on absorbance at 280 nm, and the target active component was identified by activity assay using a quality control plasma coagulation analyzer and SDS-PAGE electrophoresis.

[0161] 4. Sephadex G-75 column chromatography (column III)

[0162] Before loading the chromatography column (4.5×100cm, Shanghai Jinhua glass chromatography column), equilibrate three column volumes with 0.1M phosphate buffer (pH 7.4) at a flow rate of 2 ml / min. Check that the bacterial endotoxin level in the bottom solution does not exceed 0.25 EU / ml before loading. Take the affinity chromatography active fraction and concentrate it using a dialysis bag with a molecular weight cutoff of 10k, with polyethylene glycol as the reverse osmosis solvent. Elute with the above buffer at a rate of 2 ml / min. Detect the eluent with a UV detector and collect the protein peak fraction containing hemocoagulase.

[0163] Results Analysis

[0164] 1. Purity testing

[0165] Electrophoresis and reversed-phase high-performance liquid chromatography (Shimadzu HPLC 20A, C4 column) were used for detection. SDS-PAGE denaturing non-reducing electrophoresis detected the target protein component, showing two bands (e.g., ...). Figure 5 As shown in the figure, RP-HPLC detection showed multiple peaks, and the hemocoagulase content was only 93.7% according to the area normalization method.

[0166] 2. Target protein activity detection

[0167] The total activity of hemocoagulase in the solution after dissolving 80g of white-browed viper venom was 1.25 million units, as determined by a quality control plasma coagulation analyzer. After chromatographic purification, the collected hemocoagulase activity was 192,500 units, with a yield of 15.4%.

[0168] 3. Detection of protein content of target components

[0169] The hemocoagulase was tested using the Folin-Ciocalteu method (Chinese Pharmacopoeia, Part IV, General Chapter 0731, Protein Content Determination Method), and the protein content was 178 mg with a specific activity of 1887 units / mg.

[0170] 4. See Table 3 for a comparison of the specific results.

[0171] Table 3 Results of hemagglutinin extraction from *Viper sibirica* and *Viper lancetus* using the method of this invention.

[0172]

[0173] 5. Conclusion

[0174] The method of this invention was used to extract hemocoagulase from the venom of the Changbai Mountain white-browed viper. The hemocoagulase yield was only 15.4%, the purity was 93.7%, and the specific activity was 1081 units / mg. All indicators were lower than those of the lancehead viper venom hemocoagulase. This patented method is particularly suitable for the extraction of lancehead viper venom hemocoagulase. Furthermore, the hemocoagulase content extracted using this method is higher, making it more suitable for large-scale production.

[0175] Comparative Example 2: Extraction of hemagglutinin from lancelet venom using existing methods

[0176] Extraction steps (Refer to Chinese patent application number CN200710099163.X)

[0177] 1. Snake venom pretreatment

[0178] Accurately weigh 10g of lancehead viper venom, dissolve it in pH 7.5, 0.05M Tris-HCl buffer to a concentration of 250mg / ml, centrifuge at 3000rpm for 10min, and collect the supernatant.

[0179] 2. DEAE Sephadex A-50 ion exchange chromatography (column I)

[0180] Before loading the chromatography column (5×100cm, Shanghai Jinhua glass chromatography column), equilibrate to three column volumes with pH 7.5, 0.05M Tris-HCl buffer at a flow rate of 1 ml / min. Load the supernatant from step 1 onto the column, elute with the above buffer for two column volumes, and then perform a linear gradient elution with 1000 ml of pH 7.5, 0.05M Tris-HCl buffer and an equal volume of pH 7.5, 0.05M Tris-HCl buffer containing 0.75M NaCl at a flow rate of 1 ml / min. Collect the protein peak based on the absorbance at 280 nm.

[0181] 3. Sephadex G-15 chromatography for salt removal (column II)

[0182] A chromatographic column (5×100cm, Shanghai Jinhua glass chromatography column) was used. The collected eluted fractions were analyzed for activity using a quality control plasma coagulation analyzer (patent CN103305591A) and identified by SDS-PAGE (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0541) electrophoresis. Target fractions with a hemocoagulase activity of ≥100 units / ml and a protein molecular weight of 36000±5000 Da were collected. Before loading, three column volumes were equilibrated with water for injection. The collected target fractions were then loaded at a flow rate of 1 ml / min. Eluting with water for injection was performed, and fractions with coagulation activity were collected using the same method.

[0183] 4. DEAE Sephadex A-50 ion exchange re-chromatography (column III)

[0184] Adjust the pH of the coagulation-active collection solution collected in step 3 to 5.2, and then perform re-chromatography. Before chromatography, equilibrate three column volumes with 0.05M Tris-HCl at pH 5.2. Load the collected target fraction at a flow rate of 1 ml / min, and then perform linear gradient elution with 1000 ml of 0.05M Tris-HCl buffer at pH 5.2 and an equal volume of 0.25M Tris-HCl buffer containing NaCl at pH 5.2 and an equal volume of 0.25M Tris-HCl buffer at pH 5.2 and an equal volume of 0.05M Tris-HCl buffer containing NaCl at a flow rate of 1 ml / min. Collect the coagulation-active fraction in the eluent using the same method as in step 3.

[0185] 5. Sephadex G-75 column chromatography (column IV)

[0186] Before chromatography, the chromatography column (5×100cm, Shanghai Jinhua glass chromatography column) is equilibrated with three column volumes of water for injection at a flow rate of 1ml / min. During chromatography, after the supernatant has completely moved below the gel surface, 50ml of water for injection is added above the gel surface, and then eluted with water for injection at a flow rate of 1ml / min. The coagulation-active fraction of the eluent is collected using the same method as in step 3.

[0187] Results Analysis

[0188] 1. Purity testing

[0189] Electrophoresis and reversed-phase high-performance liquid chromatography (Shimadzu HPLC 20A, C4 column) were used for detection. SDS-PAGE denaturing non-reducing electrophoresis showed the target protein component as two bands (e.g., ...). Figure 6 As shown in the figure, RP-HPLC detection showed multiple peaks, and the hemocoagulase content was only 94.2% according to the area normalization method.

[0190] 2. Target protein activity detection

[0191] The total activity of hemocoagulase in the solution after dissolving 10g of white-browed viper venom was 710,000 units, as determined by the quality control plasma coagulation analyzer method. After chromatographic purification, the collected hemocoagulase activity was 57,500 units, with a yield of 8.1%.

[0192] 3. Detection of protein content of target components

[0193] The hemocoagulase was detected by the Folin-Ciocalteu method (Chinese Pharmacopoeia, Part IV, General Chapter 0731, Protein Content Determination Method), and the protein content was 18 mg with a specific activity of 3194 units / mg.

[0194] 4. See Table 4 for a comparison of the specific results.

[0195] Table 4. Results of extracting hemagglutinin from *Lymnocypris lanceolata* using existing methods and the method of this invention.

[0196]

[0197] 5. Conclusion

[0198] Existing methods for extracting hemocoagulase from lancehead viper venom yield acceptable purity and specific activity (94.2% and 3194 units / mg, respectively), but the yield is significantly lower than the 38.2% obtained by the method of this invention, at only 8.1%. This patented method involves fewer purification steps (only three), has a shorter production cycle, and a higher yield, making it more suitable for large-scale hemocoagulase production. Simultaneously, it ensures high specific activity and avoids immunogenic reactions.

[0199] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements. sequence list <110> Liaoning Yuanda Nuokang Biopharmaceutical Co., Ltd. <120> Hemocoagulase extracted from the venom of the Brazilian spearhead viper, its preparation method, and its uses. <130> DIC16110037 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 232 <212> PRT <213> Artificial sequence <220> <223> amino acid sequence of hemocoagulase <400> 1 Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His Pro Phe Leu Ala 1 5 10 15 Phe Met Tyr Tyr Ser Pro Gln Tyr Phe Cys Gly Met Thr Leu Ile Asn 20 25 30 Gln Glu Trp Val Leu Thr Ala Ala His Cys Asp Lys Thr Tyr Met Arg 35 40 45 Ile Tyr Leu Gly Ile His Thr Arg Ser Val Ala Asn Asp Asp Glu Val 50 55 60 Ile Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Lys Asn 65 70 75 80 Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu Asn Arg Pro Val 85 90 95 Lys Asn Ser Thr His Ile Ala Pro Ile Ser Leu Pro Ser Asn Pro Pro 100 105 110 Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly Ala Ile Thr Thr 115 120 125 Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu 130 135 140 Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly Leu Pro Ala Lys 145 150 155 160 Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Gly Gly 165 170 175 Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Gln Phe Gln Gly Ile Leu 180 185 190 Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys Pro Ala Phe Tyr 195 200 205 Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser Ile Ile Ala Gly 210 215 220 Asn Lys Thr Ala Thr Cys Pro Pro 225 230

Claims

1. A method for extracting hemagglutinin from the venom of the spearhead viper, the method comprising the following steps: 1) Dissolve the venom of the spearhead viper in a Tris-HCl buffer solution at pH 7.0-7.5, 0.01M-0.05M, to obtain a solution with a concentration of 50-200 mg / ml, and then centrifuge to collect the supernatant; 2) Perform DEAE Sephadex A-25 ion exchange chromatography on the supernatant obtained in step 1). Use Tris-HCl buffer containing 0.4M NaCl, pH 7.4, and concentration 0.02M for linear elution. Collect the eluent based on the absorption peak at a wavelength of 280nm to obtain the eluent containing coagulation active components. 3) The eluent containing the coagulation active component obtained in step 2) was dialyzed against a Tris-HCl buffer solution containing 1.0 M NaCl, pH 7.0, and concentration 0.05 M, and then subjected to Benzamidine Sepharose 4FF (HS) affinity chromatography, eluted with a Tris-HCl buffer solution containing 0.1 M arginine, 1.0 M NaCl, pH 7.0, and concentration 0.05 M, and the target component was collected. 4) The target component collected in step 3) was subjected to Sephadex G-75 gel filtration chromatography, eluted with 0.1M phosphate buffer at pH 7.4, and the eluent was collected to obtain the stock solution of lancehead viper venom hemocoagulase.

2. The method according to claim 1, characterized in that, In step 1), a solution with a concentration of 80-150 mg / m³ is obtained.

3. The method according to claim 1, characterized in that, In step 1), a solution with a concentration of 100 mg / ml is obtained.

4. The method according to claim 1, characterized in that, In step 1), the Tris-HCl buffer solution has a pH of 7.4 and a concentration of 0.02 M.

5. The method according to claim 1, characterized in that, In step 1), the centrifugation conditions are: 4°C, 3000 rpm-5000 rpm, and centrifugation time of 5-20 min.

6. The method according to claim 5, characterized in that, In step 1), the centrifugation time is 10 min.

7. The method according to claim 1, characterized in that, In step 2), before performing DEAE Sephadex A-25 ion exchange chromatography, three column volumes were equilibrated with Tris-HCl buffer at pH 7.0-7.5 and a concentration of 0.01M-0.05M.

8. The method according to claim 7, characterized in that, The column flow rate is 0.8-3 ml / min.

9. The method according to claim 7, characterized in that, The column flow rate was 2 ml / min.

10. The method according to claim 7, characterized in that, The Tris-HCl buffer solution has a pH of 7.4 and a concentration of 0.02 M.

11. The method according to claim 1, characterized in that, In step 2), the eluent containing the coagulation active component is tested for activity using a plasma coagulation analyzer and the eluent containing the coagulation active component is tested using SDS-PAGE electrophoresis to identify the target component.

12. The method according to claim 1, characterized in that, In step 3), before performing Benzamidine Sepharose 4FF (HS) affinity chromatography, dialyze the eluent containing 0.5M-1.5M NaCl, pH 7.0-7.5, and concentration 0.01-0.06M, and sterilely dialyze the eluent containing the coagulation active component through an ultrafiltration membrane 2-3 times.

13. The method according to claim 12, characterized in that, The Tris-HCl buffer solution has a pH of 7.0 and a concentration of 0.05 M.

14. The method according to claim 12, characterized in that, The Tris-HCl buffer solution contains 1.0 M NaCl.

15. The method according to claim 12, characterized in that, The ultrafiltration membrane is an ultrafiltration membrane package.

16. The method according to claim 12, characterized in that, The ultrafiltration membrane has a molecular weight cutoff of 10K.

17. The method according to claim 12, characterized in that, The ultrafiltration membrane is made of polyethersulfone.

18. The method according to claim 1, characterized in that, In step 3), before using the Benzamidine Sepharose 4FF (HS) affinity chromatography, equilibrate three column volumes with Tris-HCl buffer containing 0.5 M-1.5 M NaCl, at a pH of 7.0-7.5 and a concentration of 0.01-0.06 M.

19. The method according to claim 18, characterized in that, The column flow rate is 1.0 ml - 3 ml / min.

20. The method according to claim 18, characterized in that, The column flow rate was 2 ml / min.

21. The method according to claim 18, characterized in that, The Tris-HCl buffer solution has a pH of 7.0 and a concentration of 0.05 M.

22. The method according to claim 18, characterized in that, The Tris-HCl buffer solution contains 1.0 M NaCl.

23. The method according to claim 1, characterized in that, In step 4), before performing Sephadex G-75 gel filtration chromatography, equilibrate three column volumes with phosphate buffer at pH 7.0-7.5 and a concentration of 0.05M-0.3M.

24. The method according to claim 23, characterized in that, The column flow rate is 0.8-3 ml / min.

25. The method according to claim 23, characterized in that, The column flow rate was 2 ml / min.

26. The method according to claim 23, characterized in that, The phosphate buffer solution has a pH of 7.4 and a concentration of 0.1M.

27. The method according to claim 1, characterized in that, In step 4), after chromatography, the eluent is concentrated using a dialysis bag with a molecular weight cutoff of 10k.

28. The method according to claim 27, characterized in that, Polyethylene glycol is used as a reverse osmosis agent.

29. The method according to any one of claims 1-28, characterized in that, The method further includes filtering, dispensing, and freeze-drying the obtained lancehead viper venom hemocoagulase stock solution to obtain lancehead viper venom hemocoagulase freeze-dried powder.

30. The method according to claim 29, characterized in that, The lyophilized powder of *Viper lanceolata* venom hemocoagulase was prepared together with excipients and a protective agent to obtain an injectable *Viper lanceolata* venom hemocoagulase preparation.

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