Method for separating and purifying hemocoagulase from snake venom of Agkistrodon halys at Changbai Mountain

Abstract

The invention discloses a method for separating and purifying hemocoagulase from snake venom of agkistrodon halys at Changbai Mountain. The method comprises the following steps: sequentially carrying out CAPTO SP ImpRes cation column chromatography and SOURCE 15 PHE hydrophobic column chromatography on a Changbai Mountain agkistrodon halys venom solution, so as to obtain the hemocoagulase. According to the method disclosed by the invention, purification is carried out by adopting different purification principles of cation chromatography and hydrophobic chromatography so as to remove impure protein residues; the problem of few impure protein residues is solved; by improving the chromatography technology and conditions, the use of precipitation, dialysis and other technologies is avoided, and next chromatography is carried out by adjusting the conductivity, the pH value and the like, so that the activity loss is reduced, and the production period is shortened; the method provided by the invention has the advantages of large one-time chromatography sample loading amount, high yield and high purity, and is beneficial to industrial production.

Description

Technical Field

[0001] The present invention relates to a method for separating and purifying hemocoagulase from snake venom, and particularly to a method for separating and purifying hemocoagulase from the venom of Gloydius blomhoffii brevicaudus from Changbai Mountain. Background Art

[0002] In 1936, Austrian scholars Klobusitzky and Konig first isolated a thrombin-like enzyme (TLE) with procoagulant properties from the venom of Bothrops atrox using ammonium sulfate and lead acetate precipitation methods and dialysis. It was first named hemocoagulase and later named batroxobin by the World Health Organization. Twenty years later, with the gradual maturity of quality standards and protein separation techniques, an injectable hemocoagulase for hemostasis research, blood coagulation, diagnosis, and treatment made from hemocoagulase came out. Due to the high application value of hemocoagulase, many companies have invested a large amount of manpower and material resources in the development and research of TLE. However, due to the complex composition of snake venom and the low content of TLE in snake venom proteins, it is a relatively difficult task to separate and purify TLE from snake venom. As separation and purification means, generally, methods such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, salting out, ultrafiltration, and freeze-drying are required to obtain homogeneous TLE. The molecular weights and isoelectric points of TLE from different species of snake venom vary, so the methods for separating and purifying TLE from different species of snake venom are not fixed, and different purification modes need to be determined according to their respective characteristics. Currently, reports on the separation and purification of TLE from various snake venoms have been made at home and abroad.

[0003] In the prior art, when dealing with large-scale snake venom, the one-time sample loading amount is small, the separation effect is poor, and it cannot meet the requirements of industrial production. Secondly, the recovery rate of the prior art is low, which not only increases the production cost but also limits its application in industrial production. Thirdly, it is difficult to prepare a protein of high purity and single component or an active component protein in the prior art, which limits the application range and use effect of hemocoagulase. There are certain side effects in clinical applications. Finally, when separating and purifying hemocoagulase in the prior art, anion exchange chromatography, molecular sieve chromatography, and affinity chromatography methods are often used. Using these purification processes, there are problems such as long production cycle, low recovery rate, and low purity. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for separating and purifying hemocoagulase from the venom of Gloydius blomhoffii brevicaudus from Changbai Mountain, which has a large sample loading amount per chromatography, high recovery rate, and high purity, and is conducive to industrial production.

[0005] The present invention first provides a method for separating and purifying hemocoagulase from the venom of Gloydius blomhoffii brevicaudus from Changbai Mountain, comprising the following steps: The venom solution of Gloydius blomhoffii brevicaudus from Changbai Mountain was successively subjected to CAPTO SP ImpRes cation exchange column chromatography and SOURCE15 PHE hydrophobic column chromatography to obtain hemocoagulase.

[0006] In the above method, the concentration of the venom solution of Gloydius blomhoffii brevicaudus from Changbai Mountain is 50 - 200 g / L; The solvent of the venom solution of Gloydius blomhoffii brevicaudus from Changbai Mountain is Tris-HCl buffer with a pH of 7.5 - 8.5 and a concentration of 0.02 - 0.05 mol / L; specifically, it is Tris-HCl buffer with a pH of 8.0 and a concentration of 0.05 mol / L.

[0007] In the above method, the steps of the CAPTO SP ImpRes cation exchange column chromatography include the following: loading the venom solution of Gloydius blomhoffii brevicaudus from Changbai Mountain onto the CAPTO SP ImpRes cation exchange column; using Tris-HCl buffer with a pH of 7.5 - 8.5 and a concentration of 0.02 - 0.05 mol / L as phase A, and using Tris-HCl buffer with a pH of 7.5 - 8.5, a concentration of 0.02 - 0.05 mol / L, and a NaCl concentration of 0.5 - 1 mol / L as phase B for gradient elution, and collecting the fraction of the fourth peak in the chromatogram; Specifically, the collection of the fraction of the fourth peak is carried out at 4°C; The flow rate for loading the venom solution of Gloydius blomhoffii brevicaudus from Changbai Mountain can be 150 - 250 mL / min; The flow rate in the CAPTO SP ImpRes cation exchange column chromatography can be 150 - 250 mL / min; The conditions for the gradient elution are as follows: 0 - 15%B for 2 CV, 15 - 20%B for 3 CV, 20 - 100%B for 2 CV, 100%B for 1 CV, 100% - 0%B for 1 CV, 0%B for 1 CV.

[0008] The CAPTO SP ImpRes cation exchange column is equilibrated with Tris-HCl buffer with a pH of 7.5 - 8.5 and a concentration of 0.02 - 0.05 mol / L; Before the gradient elution, it is first rinsed with Tris-HCl buffer with a pH of 7.5 - 8.5 and a concentration of 0.02 - 0.05 mol / L; specifically, the flow rate for rinsing is 150 - 250 mL / min; the column volume for rinsing is 1 - 3 CV.

[0009] In the above method, the steps of the SOURCE 15 PHE hydrophobic column chromatography are as follows: NaCl is added to the fraction of the fourth peak obtained from the CAPTO SPImpRes cation exchange column chromatography to make the conductivity of the active component reach 120 - 140 ms / cm; then it is loaded onto the SOURCE 15 PHE hydrophobic column, with a Tris-HCl buffer solution of pH 7.5 - 8.5 and a NaCl concentration of 1 - 2 mol / L and a concentration of 0.02 - 0.05 mol / L as phase A, and a Tris-HCl buffer solution of pH 7.5 - 8.5 and a concentration of 0.02 - 0.05 mol / L as phase B for gradient elution, and the fraction of the second peak in the chromatogram is collected to obtain the hemocoagulase; Specifically, the collection of the fraction of the second peak is carried out at 4°C; The loading flow rate of the SOURCE 15 PHE hydrophobic column chromatography can be 50 - 150 mL / min; The flow rate of the gradient elution can be 50 - 150 mL / min; The conditions of the gradient elution are as follows: 0 - 100%B for 20 CV.

[0010] The SOURCE 15 PHE hydrophobic column is equilibrated with a Tris-HCl buffer solution of pH 7.5 - 8.5 and a NaCl concentration of 1 - 2 mol / L and a concentration of 0.02 - 0.05 mol / L; Before the gradient elution, it is first rinsed with a Tris-HCl buffer solution of pH 7.5 - 8.5 and a NaCl concentration of 1 - 2 mol / L and a concentration of 0.02 - 0.05 mol / L; specifically, the rinsing flow rate is 50 - 150 mL / min; the column volume of the rinsing is 1 - 5 CV.

[0011] In the above method, the method further includes the steps of sequentially using Sephadex G-25 gel filtration column chromatography and SOURCE 15 RPC reverse phase column chromatography.

[0012] In the above method, the steps of the Sephadex G-25 gel filtration column chromatography are as follows: the hemocoagulase prepared by the above method is loaded onto the Sephadex G-25 gel filtration column and eluted with water, and the active component is collected; Specifically, the collection of the active component is carried out at 4°C; The loading flow rate of the hemocoagulase can be 150 - 500 mL / min; The flow rate of the water elution can be 150 - 500 mL / min; the volume of the water elution is 0.8 - 1.5 CV.

[0013] In the above method, the steps of the SOURCE 15 RPC reversed-phase column chromatography include the following: loading the active components obtained after Sephadex G-25 gel filtration column chromatography onto the SOURCE 15 RPC reversed-phase column, and then using an aqueous solution of 0-20% acetonitrile containing 0.1% trifluoroacetic acid as phase A and an aqueous solution of 80-100% acetonitrile containing 0.1% trifluoroacetic acid as phase B for gradient elution, collecting by peaks to obtain hemocoagulase A and hemocoagulase B; Specifically, hemocoagulase A and hemocoagulase B are collected at 4°C; The loading flow rate of the active components obtained after Sephadex G-25 gel filtration column chromatography can be 10-50 mL / min; The flow rate of the gradient elution can be 10-50 mL / min; The conditions of the gradient elution are as follows: 0-30% B for 5 CV, 30-35% B for 10 CV, 35-100% B for 5 CV, 100-0% B for 2 CV.

[0014] The SOURCE 15 RPC reversed-phase column is equilibrated with an aqueous solution of 0-10% acetonitrile containing 0.1% trifluoroacetic acid; Before the gradient elution, it is first rinsed with an aqueous solution of 0-10% acetonitrile containing 0.1% trifluoroacetic acid; specifically, the rinsing flow rate is 10-50 mL / min; the column volume for rinsing is 1-5 CV.

[0015] The present invention also provides the hemocoagulase prepared by the above method.

[0016] And hemocoagulase A and hemocoagulase B prepared by the above method.

[0017] Finally, the present invention provides a hemocoagulase A, whose amino acid sequence is as shown in amino acid sequence 1; Specifically, the molecular weight of hemocoagulase A is 26447 Da and it contains 3 N-glycosylation sites; The present invention also provides a hemocoagulase B, whose amino acid sequence is as shown in amino acid sequence 2; Specifically, the molecular weight of hemocoagulase B is 26446 Da and it contains 2 N-glycosylation sites.

[0018] The present invention has the following beneficial effects: (1) The present invention uses a Capto SP ImpRes cation exchange column chromatography for loading the snake venom solution; the Capto SP ImpRes strong cation exchange medium has the characteristics of high binding capacity and small particle size; (2) The method of the present invention improves the recovery rate of hemocoagulase and reduces the production cost by improving chromatography techniques and conditions, which is conducive to industrialized and multi-batch production; about 1-2 mg of hemocoagulase can be prepared from each gram of snake venom. (3) The present invention purifies by adopting different purification principles of cation chromatography and hydrophobic chromatography to remove residual impurities; the problem of residual small amounts of impurities (neurotoxins of basic proteins, thrombin-like kinases) is solved. (4) The method of the present invention improves chromatography techniques and conditions, avoids using techniques such as precipitation and dialysis, and performs the next chromatography by adjusting methods such as conductivity and pH value, reducing activity loss and shortening the production cycle. (5) In the method of the present invention, the chromatography process is carried out under the condition of 18 °C, and sample collection is completed in a 4 °C light-proof vertical refrigerator, avoiding the problem that hemocoagulase gradually inactivates within 8-10 h in an aqueous solution at room temperature. (6) The present invention effectively separates and prepares high-purity hemocoagulase by adopting SOURCE 15 RPC reversed-phase chromatography column chromatography; SOURCE 15 RPC has characteristics such as a wide pH value range (1-12), excellent selectivity, chemical resistance, high load capacity, and high resolution at high flow rates; SOURCE 15 RPC is based on rigid polystyrene / divinylbenzene beads with a single diameter of 15 µm; this matrix has excellent selectivity; the monodispersity of the beads can generate a stable column bed, low back pressure, and excellent separation results at high flow rates; SOURCE 15 RPC has high physical and chemical stability and extremely high reproducibility between batches, and is very suitable for all stages of industrial-scale operation - from research and process development to scale-up and production. (7) The present invention determines the amino acid sequence for the first time. Description of the Drawings

[0019] Figure 1 It is the chromatogram of CAPTO SP ImpRes cation column chromatography; Figure 2 It is the electrophoresis diagram of the sample of CAPTO SP ImpRes cation column chromatography; Figure 3 It is the chromatogram of SOURCE 15 PHE hydrophobic column chromatography; Figure 4 It is the electrophoresis diagram of the sample of SOURCE 15 PHE hydrophobic column chromatography; Figure 5 It is the chromatogram of Sephadex G-25 gel filtration column chromatography; Figure 6 It is the chromatogram of SOURCE 15 RPC reversed-phase column chromatography; Figure 7 It is the electrophoresis diagram of the sample loaded and separated by SOURCE 15 RPC reversed-phase column chromatography; Figure 8 It is the liquid chromatogram of the sample loaded onto the SOURCE 15 RPC reversed-phase column chromatography; Figure 9 It is the liquid chromatogram of active component A; Figure 10 It is the liquid chromatogram of active component B; Figure 11 It is the amino acid sequence of active component A; Figure 12 It is the mass spectrum of active component A; Figure 13 It is the amino acid sequence of active component B; Figure 14 It is the mass spectrum of active B; Figure 15 It is the database search results of active components A and B. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0022] In the following examples, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0023] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0024] Definition of the "potency" unit of batroxobin in the following examples: Under the conditions of 37°C ± 0.5°C and shaking, the amount of enzyme in 0.2 mL of enzyme solution that causes 0.2 mL of the coagulation quality control plasma suspension to show white flocs in 58 - 62 seconds, calculated based on the concentration of the enzyme solution, is defined as containing 1 unit of batroxobin in every 1 mL of the enzyme solution.

[0025] The percentage of the solution involved in the following examples is volume percentage unless otherwise specified.

[0026] Example 1 1. Pretreatment First, 200 g of freeze-dried venom of Gloydius blomhoffii brevicaudus from Changbai Mountain was dissolved in a Tris-HCl buffer solution with a pH of 8.0 and a concentration of 0.05 mol / L to obtain a solution of Gloydius blomhoffii brevicaudus venom with a concentration of 100 g / L. It was centrifuged at 3000 r / min for 15 min at 4°C, and the supernatant was taken. The filtrate was obtained by filtering through a 200-mesh sieve, about 2 L, with a total potency of about 1,000,000 units. It was stored for standby at low temperature.

[0027] High-speed refrigerated centrifuge: TGL-16A, Hunan Pingfan Technology Co., Ltd. 2. Chromatography The following four chromatography steps are completed at 18°C: (1) CAPTO SP ImpRes cation exchange column chromatography Purification is carried out using an AKTA PILOT protein purifier (GE, product number: 56317181).

[0028] Dilute 2 L of the filtrate obtained after pretreatment with water to 3 L, and load it onto a CAPTO SP ImpRes chromatography column (chromatography column: BPG140×500 mm, GE, product number: 18111308; packing: CAPTO SP ImpRes, GE, product number: 17546803 (1 L), column volume CV: 2 L) that has been equilibrated with buffer A (pH 8.0, 0.05 mol / L Tris-HCl) at a flow rate of 200 mL / min. Then, wash with buffer A (pH 8.0, 0.05 mol / L Tris-HCl) at a flow rate of 200 mL / min for 2 CV to remove unadsorbed impurity proteins. Use buffer A as phase A and a buffer (pH 8.0, 0.05 mol / L Tris-HCl) with a NaCl concentration of 1 mol / L as phase B for gradient elution: 0 - 15% B for 2 CV, 15 - 20% B for 3 CV, 20 - 100% B for 2 CV, 100% B for 1 CV, 100% - 0% B for 1 CV, 0% B for 1 CV.

[0029] The gradient elution flow rate is 200 mL / min; online detection is carried out at a wavelength of 280 nm (ultraviolet absorption detection built into the purifier), and multiple absorption peaks are obtained respectively. The chromatography profile is shown in Figure 1 ... Collect by peaks in a 4°C vertical refrigerator (Aucma vertical transparent refrigerator, Aucma Co., Ltd., product number: SC-237). Detect the coagulation activity of the substances collected in each peak. After testing, the active component is in the 4th peak. After collection, it is stored at 4°C for later use (the collected active component is about 1 L, and the total activity is about 600,000 units). The electrophoresis profile of the 4th peak of the active component is shown in Figure 2 ... According to the electrophoresis diagram, this component contains multiple proteins.

[0030] The electrophoresis method uses the reducing gel electrophoresis method.

[0031] The detection method of coagulation activity is as follows: Equal volumes (specifically 1 mL) of the collected liquid preheated to 37°C and coagulation quality control plasma (quality control product for coagulation two-item detection, produced and traded by German M&C Medical Instruments Co., Ltd., product number: 11645073) are mixed and water-bathed at 37°C. The collected part that can coagulate the plasma within 30 s is the target component with coagulation activity.

[0032] (2)SOURCE 15 PHE hydrophobic column chromatography Purification is carried out using an AKTA PILOT protein purifier (GE Company, product number: 56317181).

[0033] NaCl is added to 1 L of the active component of the collected Peak IV above to make the conductivity of the active component reach 140 ms / cm, and it is loaded onto a SOURCE 15 PHE hydrophobic column [chromatography column: FINE LINE 100, GE Company, product number: 11002798; packing: SOURCE 15 PHE, GE Company, product number: 17014704 (1 L), column volume CV = 0.8 L] that has been equilibrated with Buffer A of 2 mol / L NaCl, pH 8.0, 0.05 mol / L Tris-HCl at a flow rate of 100 mL / min. Buffer A of 2 mol / L NaCl, pH 8.0, 0.05 mol / L Tris-HCl is used to wash for 2 CV at a flow rate of 100 mL / min, and then gradient elution is carried out with Buffer A as phase A and Tris-HCl buffer of pH 8.0, 0.05 mol / L as phase B, 0 - 100%B for 20 CV; flow rate: 100 mL / min, collected by peak in a 4°C vertical refrigerator, and the chromatogram is shown in Figure 3 ; Measure the biological activity of the active component in Peak II, and the electrophoresis pattern of the Peak II component is shown in Figure 4 , According to the electrophoresis diagram, it shows that this component contains two proteins with molecular weights of 41 kDa and 38 kDa respectively. After the active component is collected, it is stored at 4°C for standby. Approximately 4 L of active component is collected. The 4 L of active component is concentrated to 1.2 L using a 5 kDa membrane package (Pall Corporation, product number: OS005T12), and the total activity is about 350,000 units. 2 5 kDa membrane package (Pall Corporation, product number: OS005T12) to 1.2 L, with a total activity of approximately 350,000 units.

[0034] (3)Sephadex G-25 gel filtration column chromatography Purification is carried out using an AKTA PILOT protein purifier (GE Company, product number: 56317181).

[0035] Load 1.2 L of the thrombin activity sample obtained from the above-mentioned hydrophobic column chromatography onto a G-25 chromatography column at a flow rate of 500 mL / min [Chromatography column: BPG200×500 mm, GE Healthcare, product number: 18110311; packing material: Sephadex G-25, GE Healthcare, product number: 17003302 (500 g), column volume CV = 6 L]; elute with water for 1 CV at a flow rate of 500 mL / min, and collect the fractions according to the peaks in a vertical refrigerator at 4°C. This step is for desalting and replacing the buffer. The chromatogram is shown in Figure 5 ; 1.5 L of the active fraction was collected, with a total activity of approximately 300,000 units of the active fraction. Store at 4°C for later use after collection.

[0036] (4)SOURCE 15 RPC Reversed-phase Column Chromatography Purification was performed using an AKTA pure protein purification instrument (GE Healthcare, product number: 29046696).

[0037] Load 300 mL of the active fraction obtained from the above-mentioned gel filtration column chromatography onto a SOURCE 15 RPC reversed-phase chromatography column equilibrated with mobile phase A (10% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid) at a flow rate of 30 mL / min [Chromatography column: FineLINE pilot 35, GE Healthcare, product number: 56116252; packing material: GE Healthcare, product number: 17072702 (200 mL), column volume: 0.15 L]. Then wash with mobile phase A at a rate of 30 mL / min for 2 CV, and then perform gradient elution with mobile phase B (90% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid). Elution conditions: 0 - 30%B for 5 CV, 30 - 35%B for 10 CV, 35 - 100%B for 5 CV, 100 - 0%B for 2 CV; flow rate: 30 mL / min, and collect the fractions according to the peaks in a vertical refrigerator at 4°C; the chromatogram is shown in Figure 6 , the active fraction obtained from the above-mentioned gel filtration column chromatography was effectively separated by reversed-phase chromatography, and 250 mL of active fraction A and 250 mL of active fraction B were obtained; the electrophoresis pattern is shown in Figure 7 , the figure shows that the sample loaded onto the reversed-phase chromatography column, i.e., the active fraction obtained from the gel filtration column chromatography, contains two proteins with different molecular weights, 41 kDa and 38 kDa. After separation by reversed-phase chromatography, single active fractions of 41 kDa and 38 kDa were obtained respectively. Store the active fractions at 4°C for later use after collection.

[0038] Perform high-performance liquid chromatography (HPLC) detection on the active fraction, active fraction A, and active fraction B obtained from the gel filtration column chromatography. The specific detection conditions are as follows: High-performance liquid chromatography detection method: High-performance liquid chromatography instrument: Aglient 1260 II Chromatographic column: Agilent ZORBAX 300SB-C 18 (250×4.6 mm, 5 μm, pore size 300 Å) Column temperature: 25 °C Detection wavelength: 280 nm Flow rate: 1 mL / min Injection volume: 100 μL Mobile phase: A: 10% acetonitrile aqueous solution with 0.1% TFA; B: 90% acetonitrile aqueous solution with 0.1% TFA Gradient elution: 10 - 70% B; elution for 30 min The high-performance liquid chromatography (HPLC) chromatogram of the active fraction obtained by gel filtration column chromatography is shown in Figure 8 , and the figure shows that the sample contains component A and component B. The detection results are shown in Table 1. It can be seen from Table 1 that the active fraction obtained by gel filtration column chromatography contains 2 components, with contents of 51.629% and 48.371% respectively.

[0039] Table 1 Liquid phase detection results of the active fraction obtained by gel filtration column chromatography

[0040] The HPLC chromatogram of active component A is shown in Figure 9 , and the figure shows that active component A is a single component with a purity of 100%. The HPLC chromatogram of active component B is shown in Figure 10 , and the figure shows that active component B is a single component with a purity of 100%. The phase detection results of active components A and B are shown in Table 2. It can be seen from Table 2 that both active components A and B are single components with a purity of 100%.

[0041] Table 2 Liquid phase detection results of active components A and B

[0042] 3. Lyophilization The 250 mL of active components A and B obtained by SOURCE 15 RPC reverse-phase column chromatography were each added with water to 1 L, concentrated to 100 mL using a 0.1 m 2 5 kDa membrane package (Pall Corporation, part number: OS005T12), added with water to 1 L, and then concentrated to 100 mL, repeating 5 times. This step is used for concentration and buffer replacement. 100 mL of the concentrated active components A and B were each dispensed into 100 5 mL vials, with a filling volume of 2 mL each, for lyophilization. The purified active components A and B powders were obtained after lyophilization.

[0043] Lyophilization conditions are as follows: Vacuum freeze dryer (Shanghai Dongfulong Technology Co., Ltd.) Model: lyo-0.2 (1) Pre-freezing Place the vial containing the sample in a half-stoppered form into the freeze drying tray and then into the freeze dryer. Freeze at -40℃ for 4 hours.

[0044] (2) Freeze-drying At -40℃, open the vacuum state for 1h. The vacuum degree is <30Pa.

[0045] After 1 hour in vacuum, turn on the heating, raise the temperature to 0°C at a rate of 5°C per hour and vacuum dry for 2 hours.

[0046] Continue to raise the temperature to 20°C at a rate of 5°C per hour and vacuum dry for 2 hours.

[0047] After freeze-drying, the purified active components A and B powders are obtained.

[0048] After the above four steps of chromatography, two active components A and B can be separated and purified from the snake venom.

[0049] The lyophilized powder in each vial can be reconstituted with 1 mL of purified water and then tested.

[0050] The molecular weight of active component A was 41kDa as determined by SDS-PAGE gel electrophoresis, and its purity was 100% as determined by HPLC; the total activity of each vial was approximately 400 units, the protein concentration was 0.2mg / mL, and the specific activity was approximately 2000 units / mg; the molecular weight of active component B was 38kDa as determined by SDS-PAGE gel electrophoresis, and its purity was 100% as determined by HPLC; the total activity of each vial was approximately 400 units, the protein concentration was 0.2mg / mL, and the specific activity was approximately 2000 units / mg.

[0051] After conversion, 200g of snake venom can be used to prepare active component A with a total activity of 100,000 units and a mass of 50mg; active component B with a total activity of 100,000 units and a mass of 50mg.

[0052] 4. Detection (1) Potency determination Standard solution: Take 1 vial of Agkistrodon acutus hemocoagulase standard (101 units per vial; for potency determination, China Food and Drug Inspection Institute; batch number: 140824-201501), accurately add 1 mL of water to dissolve it and quantitatively dilute it to make solutions containing approximately 0.3, 0.5, 1.0, 2.0, and 3.0 units per 1 mL, respectively.

[0053] Test solution: Take an appropriate amount of this product, melt and mix at room temperature, accurately measure an appropriate amount, and add water to quantitatively dilute to make a solution containing approximately 1.0 unit per 1 mL.

[0054] Assay: Take 0.1 mL of the coagulation quality control plasma suspension [1 mL / vial of coagulation quality control plasma (MDC Hemostasis) is placed at room temperature, 1.0 mL of water is added to each vial, left standing for 15 min, gently shaken during this period, and gently shaken well before each use], add it to 20 reaction cups respectively and place them in a coagulation analyzer, preheat at 37°C ± 0.5°C for 2 min, accurately measure 0.2 mL of each standard solution with a series of concentrations, quickly add it to the above-mentioned reaction cups, and record the clotting time. Each concentration is determined 4 times repeatedly, and the average value is calculated (the difference between the maximum and minimum values of the 4 determinations shall not exceed 10% of the average value, otherwise re-determine). With the logarithm of the standard solution concentration as the abscissa and the logarithm of the clotting time as the ordinate, calculate the linear regression equation, and the correlation coefficient should be greater than 0.99. Take the test solution, determine it according to the above method, obtain the concentration of the test solution from the regression equation, multiply it by the dilution factor, and calculate to obtain the result.

[0055] Definition of the unit of batroxobin: Under the conditions of 37°C ± 0.5°C and shaking, the amount of enzyme in 0.2 mL of enzyme solution that causes 0.2 mL of coagulation quality control plasma suspension to show white flocs in 58 - 62 seconds, calculated by the concentration of the enzyme solution, is defined as containing 1 unit of batroxobin in every 1 mL of enzyme solution.

[0056] (2)Protein content Reference substance solution: Take 1 vial of bovine serum albumin reference substance, quantitatively dilute it with water to prepare a reference substance stock solution containing about 0.5 mg per 1 mL; accurately measure 0.0 mL, 0.01 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, 0.1 mL of the reference substance stock solution respectively, place them in stoppered test tubes, and make up to 0.1 mL with water respectively as the reference substance solutions.

[0057] Test solution: Take an appropriate amount of this product, melt and mix it at room temperature, accurately measure 0.1 mL and place it in a stoppered test tube as the test solution.

[0058] Determine according to the protein content determination method (Method 5 in General Principles 0731, Volume IV, Chinese Pharmacopoeia 2020 Edition), and calculate the amount of protein (mg) per 1 mL.

[0059] Specific activity: Divide the activity unit of batroxobin in this product by the amount of protein (mg) to obtain the result.

[0060] 5. Analysis of active components (1)Amino acid sequence and molecular weight Perform mass spectrometry identification on the active components A and B.

[0061] The amino acid sequence of active component A (the red font indicates the N-glycosylation site) is shown in Figure 11。The calculated molecular weight of the amino acid sequence of active component A is 26,456 Da; after removing N-glycans with PNGase F, the molecular weight becomes 26,447 Da. The molecular weight of active component A after N-glycan removal analyzed by high-resolution mass spectrometry is 26,447 Da, and it contains 3 N-glycosylation sites, as shown in Figure 12 , which is consistent with the theoretically calculated molecular weight.

[0062] The amino acid sequence of active component B (the N-glycosylation sites are in red font) is shown in Figure 13 。After comparison, the amino acid sequences of active components A and B are exactly the same.

[0063] The calculated molecular weight of the amino acid sequence of active component B is 26,456 Da; after removing N-glycans with PNGase F, the molecular weight becomes 26,446 Da. The molecular weight of active component B after N-glycan removal analyzed by high-resolution mass spectrometry is 26,446 Da, and it contains 2 N-glycosylation sites, as shown in Figure 14 , which is consistent with the theoretically calculated molecular weight.

[0064] (2)NCBI database analysis Searching the NCBI database, it is found that both active components A and B are TLE (hemocoagulase). The protein sequence coverage rate is 61%. The sequence contains N-glycosylation sites, and it is speculated that the heterogeneity of active components A and B in electrophoresis may be caused by N-glycosylation.

[0065] The specific database search and comparison results are as follows: The database search and comparison results of active components A and B show that the highest coverage is P85109 TLE, with a coverage rate of 61%. See Figure 15 。

Claims

1. A method for separating and purifying hemocoagulase from the venom of Agkistrodon halyssopus, comprising the following steps: The venom solution of Agkistrodon halyssa from Changbai Mountain was chromatographed by CAPTO SP ImpRes cationic column and SOURCE 15PHE hydrophobic column in sequence to obtain hemocoagulase.

2. The method according to claim 1, characterized in that: The concentration of the Changbai Mountain Agkistrodon acutus venom solution is 50-200 g / L; The solvent of the Changbai Mountain Agkistrodon acutus venom solution is a Tris-HCl buffer solution with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L.

3. The method according to claim 1 or 2, characterized in that: The CAPTO SP ImpRes cationic column chromatography step comprises the following steps: loading the Changbai Mountain Agkistrodon acutus venom solution onto a CAPTO SP ImpRes cationic column; using a Tris-HCl buffer solution with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L as phase A, and using a Tris-HCl buffer solution with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L containing NaCl at a concentration of 0.5-1 mol / L as phase B, performing gradient elution, and collecting the IV peak component in the chromatogram; Specifically, the collection of the peak IV component is carried out at 4°C; The flow rate of loading the Changbai Mountain Agkistrodon acutus venom solution may be 150-250 mL / min; The flow rate in the CAPTO SP ImpRes cationic column chromatography may be 150-250 mL / min; The conditions of the gradient elution were as follows: 0-15% B 2CV, 15-20% B 3CV, 20-100% B 2CV, 100% B 1CV, 100%-0% B 1CV, 0% B 1CV; Before gradient elution, Tris-HCl buffer with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L can be used for elution.

4. The method according to any one of claims 1 to 3, characterized in that: The SOURCE 15 PHE hydrophobic column chromatography step comprises the following steps: adding NaCl to the peak component IV obtained by the CAPTO SP ImpRes cationic column chromatography to make the conductivity of the active component reach 120-140 ms / cm; then loading it onto the SOURCE 15 PHE hydrophobic column, and then using a Tris-HCl buffer solution with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L and a NaCl concentration of 1-2 mol / L as phase A, and a Tris-HCl buffer solution with a pH of 7.5-8.5 and a concentration of 0.02-0.05 mol / L as phase B, performing gradient elution, and collecting the peak component II in the chromatogram to obtain the hemocoagulase; Specifically, the peak II fraction is collected at 4°C; The sample loading flow rate of the SOURCE 15 PHE hydrophobic column chromatography can be 50-150 mL / min; The flow rate of the gradient elution may be 50-150 mL / min; The conditions of the gradient elution were as follows: 0-100% B 20CV; Before the gradient elution, the solution is firstly eluted with a Tris-HCl buffer having a pH of 7.5-8.5 and a NaCl concentration of 1-2 mol / L and 0.02-0.05 mol / L.

5. The method according to any one of claims 1 to 4, characterized in that: The method further comprises the steps of sequentially using Sephadex G-25 gel filtration column chromatography and SOURCE 15 RPC reverse phase column chromatography.

6. The method according to claim 5, characterized in that: The step of Sephadex G-25 gel filtration column chromatography comprises the following steps: loading the hemocoagulase prepared by the method according to any one of claims 1 to 4 onto a Sephadex G-25 gel filtration column, eluting with water, and collecting active components; Specifically, the active component is collected at 4°C; The loading flow rate of the hemocoagulase can be 150-500 mL / min; The flow rate of the water elution may be 150-500 mL / min.

7. The method according to claim 5 or 6, characterized in that: The steps of the SOURCE 15 RPC reverse phase column chromatography are as follows: the active component obtained after the Sephadex G-25 gel filtration column chromatography is loaded onto the SOURCE 15 RPC reverse phase column, and then 0.1% trifluoroacetic acid in a 0-10% acetonitrile aqueous solution is used as phase A, and 0.1% trifluoroacetic acid in a 80-100% acetonitrile aqueous solution is used as phase B, and gradient elution is performed, and peaks are collected to obtain hemocoagulase A and hemocoagulase B; Specifically, the hemocoagulase A and hemocoagulase B are collected at 4°C; The loading flow rate of the active component obtained after the Sephadex G-25 gel filtration column chromatography can be 10-50 mL / min; The flow rate of the gradient elution can be 10-50 mL / min; The conditions of the gradient elution were as follows: 0-30% B 5CV, 30-35% B 10CV, 35-100% B 5CV, 100-0% B 2CV; The gradient elution was preceded by elution with a 0-10% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid.

8. The hemocoagulase prepared by the method according to any one of claims 1 to 4.

9. Hemocoagulase A and hemocoagulase B prepared by the method according to any one of claims 5 to 7.

10. A hemocoagulase A, characterized in that: The amino acid sequence of the hemocoagulase A is as shown in amino acid sequence 1; Specifically, the molecular weight of the hemocoagulase A is 26447 Da; A hemocoagulase B, characterized in that: The amino acid sequence of the hemocoagulase B is as shown in amino acid sequence 2; specifically, the molecular weight of the hemocoagulase B is 26446 Da.

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