CN109207461B

CN109207461B - Blood coagulation factor X activator and preparation method thereof

Info

Publication number: CN109207461B
Application number: CN201710550953.9A
Authority: CN
Inventors: 丁忠福; 李秀琳; 李秀娜; 崔亮亮; 李香南; 李萍; 薛雁; 王宏英; 薛百忠
Original assignee: Yuanda Life Science Liaoning Co ltd
Current assignee: Yuanda Life Science Liaoning Co ltd
Priority date: 2017-07-07
Filing date: 2017-07-07
Publication date: 2021-09-28
Anticipated expiration: 2037-07-07
Also published as: CN109207461A

Abstract

The invention provides a blood coagulation factor X activator, which is derived from agkistrodon spearhead (Bothrops Atrox) snake venom, and a preparation method thereof. The invention also provides application of the blood coagulation factor X activator in preparing a medicament for treating bleeding diseases and a pharmaceutical composition for treating bleeding. Compared with the prior art, the coagulation factor X activator provided by the invention has high purity, the purity is 100% by using a Size Exclusion Chromatography (SEC) column for detection, and the purity is up to 96% by using a C4 reverse phase column for detection. The method provided by the invention firstly uses anion-cation exchange filler chromatography, and then carries out affinity chromatography or composite filler chromatography, so that the purity of the blood coagulation factor X activator after the affinity column chromatography is close to that of electrophoresis, the service life of the affinity filler is greatly prolonged, the production cost is saved, and the method is favorable for large-scale industrial production.

Description

Blood coagulation factor X activator and preparation method thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a blood coagulation factor X activator derived from venom of Bothrops atrox, a preparation method of the activator and application of the activator.

Background

Snake venom is rich in various proteases and polypeptides acting on various links of the blood coagulation system of mammals, and the components have specificity on blood coagulation factors and are widely applied to the aspects of anticoagulation, antithrombotic, hemostasis and the like. The Factor X Activator (FXA) specifically acts on the coagulation Factor X (FX), and enables the FX to be rapidly converted into the activated coagulation factor X (FXa), prothrombin is activated under the action of the FXa to form active thrombin, and the thrombin acts on plasma fibrinogen to convert the plasma fibrinogen into fibrin so as to play a role in stopping bleeding. The blood coagulation factor X activator obtained from snake venom is more effective than the existing hemostatic, and has great application value in clinical operation hemostasis. F XA is widely distributed in snake venom of Viperaceae, Agkistrodon, Elapidae, etc.

Separation of hemostatic from snake venom has a long history, and at present, there is a successful clinical application of Reptilase (Chinese trade name: Haemorrhagic) produced in Switzerland, which is a mixture of thrombin-like enzyme extracted from venom of Agkistrodon halys boviensis and a small amount of activator of coagulation factor X. A domestic hemostatic imitation-batroxobin is also an enzyme hemostatic prepared from batroxobin extracted from venom of Bothrops atrox. In China, the blood coagulation factor X activator is also used as a viper venom kit for diagnosing whether blood plasma is lack of the blood coagulation factor X.

The research on the snake venom blood coagulation factor X activator is reported at home and abroad, the most extensive and most intensive research is on viper venom, such as the Vipera perla produced in Tai, and the like, the Vipera perla venom is taken as a raw material, CM-Sephadex C-50 (weak cation exchange filler) → Sephadex G-75 → Superdex 75 is adopted for three-step purification, separation and purification to obtain an F X activator VRS-X, under the reducing and non-reducing conditions, the VRS-X is determined to be a peptide chain through SDS-PAGE, and the molecular weight is determined to be 61.1 +/-1.5 KDa, 60.4 +/-1.9 KDa (detailed in the Vipera perla et al, Vipera russelii Sidemia) purification and partial characteristic research of the snake venom factor X activator [ J ] Fujian medical science report, 2002, 36 (3): 246-. Xiyun and the like use Guangxi Vipera viper Venom as a raw material, and sequentially carry out two-step chromatographic separation and purification by DEAE FF → Superde X G75 to obtain an FX activator (factor X activator of Russell' S sight Venom, RVV-X), which is a single protein band under the non-reducing condition of SDS-PAGE, has the molecular weight of about 97.8kDa, is a two-protein band under the reducing condition, and is a heavy chain (about 76.3kDa) and a light chain (about 21.5kDa) (Xiyun, Schlulu, the research on the separation and purification and the hemostatic action of the Guangxi Viper viper Venom factor X activator [ J ] Chinese practical medicine, 2010, 5 (24): 9-11). In 1976 Furukawa et al obtained RVV-X from viper venom (Russell's snake) by Sephadex G-50 → DEAE cell → Sephadex G-200 three-step chromatography in sequence, which showed four protein bands under SDS-PAGE reducing conditions, 61000, 19800, 16500 and 13000Da (Furukawa, Y., Matsunaga, Y., Hayashi, K. (1976). Purification and characterization of a complex protein from the snake venom of Russell's virus. Biochim. biophysis.acta 453:48-61), respectively. Kisiel et al obtained RVV-X from viper venom (Russell's viper venom) using Sephadex G-150 and QAE-Sephadex A-50 two-step chromatography, which under SDS-PAGE reducing conditions gave three protein bands, 5900, 20000 and 18000Da (Kisiel, W., Hermodson, M.A., Davie, E.W, (1976). Factor X activating enzyme from Russell's viper venom: isolation and chromatography. biochemistry15(22): 4901): 4906).

Related patents related to separation of snake venom blood coagulation factor X activator (RVV-X) from viper venom in China, such as the Chinese patent invention CN02153855 named as 'chromatography purification method of viper venom blood coagulation enzyme' discloses that the purification process of RVV-X is 3-step chromatography, and then Heparin Sepharose CL-6B (Heparin affinity column), DEAE-Sepharose Fast Flow (anion exchange) and Sephacryl-300HR (gel filtration) fillers are sequentially used. However, in the method, because the ingredients of snake venom raw materials are complex, the affinity column is used for damaging the filler in the first step, and Sephacryl-300HR (gel filtration) filler is used in the third step, and the filler belongs to a soft rubber filler, so that the pressure resistance is weak, the flow rate is slow in the chromatography process, and further production cannot be amplified.

Chinese invention patent 03101881.5 entitled "a hemostatic" discloses a purification method of RVV-x by using two steps of crude fractionation with Sephacryl series gel filtration column and Q-Sepharose series ion exchange column, although snake venom source is not described in the application, it can be presumed that the snake venom source is vipers based on the disclosed snake venom coagulation factor x activator (RVV-x).

The invention discloses a technical scheme for RVV-X purification, which is disclosed in Chinese patent application No. 98119978.X entitled "method for efficiently extracting blood coagulation factor decactivator", and an electrophoresis pure sample is obtained from viper venom by one-step chromatography through a Mono Q column (anion exchange column).

Chinese patent application 200710102028.6 entitled Vipera peruvii venom blood coagulation X factor activating enzyme and extraction method and application thereof discloses a technical scheme for purifying RVV-X, which is prepared from Vipera peruvii venom by sequentially using DEAE-Sephadex A-50 anion exchange chromatography, cellulose DEAE-32 anion exchange chromatography and Superose-12 gel filtration chromatography. The cellulose DEAE-32 anion exchange filler used in the method is soft gel, and the column volume of the filler can change along with the change of the ionic strength in the buffer solution, so the method has poor controllability of the chromatography process and is not beneficial to large-scale production.

Chinese patent 200410020115.3 entitled "a compound biological component with procoagulant action and its pharmaceutical preparation" discloses a method for separating and purifying thrombin and thromboplastin (blood coagulation factor ten activating enzyme), the purification process is Sephadex G-25 (gel filtration column) chromatography, then QAE-Sephadex A50 (anion exchange column) chromatography, molecular sieve gel filler for the first step of chromatography, the method has the disadvantages of severely limiting the loading amount of snake venom, and the chromatography process consumes a long time and can not be further amplified for production.

While relatively few studies have been made on activators of coagulation factor X from Bothrops atrox, Hofmann et al, by DEAE cellulose → Freeze-drying dialysis → Sephadex G-100 steps, obtained activator of F X (Hofmann, H., Dumarey, C., Bon, C. (1983), Blood clotting by Bothrops atrox vehicle identification and properties of a factor X activator. Biochimie 65(3): 201-210). The invention is named as 'a compound hemostatic originated from snake venom', the snake venom source of F XA separated and purified in Chinese patent application CN200510047332 is from Bothrops atrox venom, and the purification is realized by DEAE-Fast Flow (anion exchange filler) and Sephacryl series (gel filtration filler) filler. Apparently, there is currently a lack of research to obtain factor x activators from agkistrodon spearhead.

In summary, the raw materials for purifying the current blood coagulation factor X activator are mainly separated and purified from viper venom, and the preparation method is complicated, so that the production cost is greatly increased, and the purification method cannot be used for large-scale industrial production.

Thus, there is a need for new factor x activators and more controlled methods of preparation.

Disclosure of Invention

Therefore, the present invention aims to overcome the defects of the prior art and provide a factor X activator and a preparation method thereof. The coagulation factor X activator provided by the invention has high purity, is detected by a size exclusion chromatography SEC column, has the purity of 100 percent, is detected by a C4 reverse phase column, has the purity of 96 percent, can be widely used for preparing medicines for treating clinical bleeding and hemorrhagic diseases such as surgical bleeding, internal medicine bleeding, gynecological bleeding, facial bleeding, pediatric bleeding and bleeding after tissue biopsy, can play an important role in the field of biological pharmacy, has wide application prospect, and can be produced in a large scale due to controllable process of the preparation method of the coagulation factor X activator.

In one aspect, the invention provides a factor X activator derived from venom of Bothrops Atrox, wherein the factor X activator has an isoelectric point of 4.94-6.64; in non-reducing SDS-PAGE, said factor X activator is in a single band and has a measured molecular weight of 70-96kDa, more preferably 75-88 kDa; in reducing SDS-PAGE, the factor x activator is in three bands, chain H, chain L1, and chain L2, and the molecular weights determined are chain H: 50-64 kDa; chain L1: 11-16 kDa; chain L2 of 11-16 kDa;

preferably, the N-terminal sequence of the H chain of the factor X activator is as shown in SEQ ID NO: 1: 1 LTPEQQAYLDAKKYV;

preferably, the N-terminus of the L1 chain of the factor X activator is as shown in SEQ ID NO: 2:

SEQ ID NO:2DCPSDWSSYEGHCYR；

preferably, the N-terminal sequence of chain L2 of the factor X activator is as shown in SEQ ID NO: 3:

SEQ ID NO:3DCNSYWSTYEGRSYR；

preferably, the glycosylase cleavage of the factor X activator shows a sugar site in the heavy chain that is an N-sugar; preferably, the N-glycosylation content is in the range of 25 to 34%, more preferably 27 to 33%, still more preferably 28.8% to 31.7% by MALDI-TOF assay;

preferably, the molecular weight of the factor X activator is 85-90kDa, more preferably 86-88kDa, even more preferably 87.4kDa-87.6kDa, as determined by MALDI-TOF assay;

preferably, the ESI-Q-TOF assay determines the molecular weight of the factor X activator to be 84-90 KDa; more preferably 86-89kDa, even more preferably 85.7kDa-88.2 kDa;

preferably, the desugarized molecular weight of the factor X activator is 59-62kDa, more preferably 61kDa, as determined by MALDI-TOF;

preferably, the molecular weight of the chain of L1 as determined by ESI-Q-TOF is from 13 to 16kDa, more preferably from 14 to 15kDa, still more preferably from 14.7 to 14.8 kDa;

preferably, the molecular weight of the chain of L2 as determined by ESI-Q-TOF is from 13 to 16kDa, more preferably from 14 to 15kDa, even more preferably 14.5 kDa;

preferably, the molecular weight of the H chain is 52-64kDa, more preferably 55-62kDa, as determined by ESI-Q-TOF.

In another aspect, the present invention provides a method of preparing a factor x activator, the method comprising the steps of:

dissolving venom of Agkistrodon halys, centrifuging, collecting supernatant, and sequentially performing anion exchange chromatography, cation exchange chromatography, affinity or composite filler chromatography, anion exchange chromatography, and gel filtration chromatography to obtain factor X activator.

Preferably, the method of the present invention comprises the steps of:

1) dissolving Agkistrodon halys venom in 0.01-0.1mol/L alkaline buffer solution with pH of 7.1-9.0 to obtain solution with concentration of 20-150mg/ml, centrifuging, and collecting supernatant;

2) carrying out anion exchange chromatography and linear gradient elution on the supernatant obtained in the step 1), collecting eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 5000-30000 kDa and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an alkaline buffer solution with the pH of 7.0-8.9 and the concentration of 0.01-0.1 mol/L; the gradient eluent B is an alkaline buffer solution which contains 0.1-0.6mol/L NaCl, has the pH value of 7.0-8.9 and has the concentration of 0.01-0.1 mol/L;

wherein the dialysate is acidic buffer solution with pH of 5.0-6.8 and concentration of 0.02-0.1 mol/L;

preferably, the filler for anion exchange chromatography is selected from fillers whose filler ligands are of the quaternary amine, tertiary amine, secondary amine and primary amine group type; more preferably, the anion exchange chromatography packing is DEAE-Sepharose Fast Flow;

preferably, the dialyzed eluate is stored at 4 ℃;

3) performing cation exchange chromatography and linear gradient elution on the eluent dialyzed in the step 2), collecting the eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 5000-30000 kDa and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an acid buffer solution with pH of 5.0-6.8 and concentration of 0.02-0.1mol/L, and the gradient eluent B is an acid buffer solution with pH of 5.0-6.8 and concentration of 0.02-0.1mol/L, which contains 0.1-0.6mol/L NaCl;

preferably, the dialysis liquid is selected from affinity chromatography dialysis liquid or composite filler chromatography dialysis liquid corresponding to the next step of affinity chromatography or composite filler chromatography; more preferably, the affinity chromatography dialysate is a dialysate containing 0-2mol/L NaCl, 0.001-0.005mol/L CaCl₂And MnCl₂An alkaline buffer solution with pH of 7.0-8.5 and concentration of 0.01-0.1 mol/L; the chromatography dialysate of the composite filler is an alkaline buffer solution which contains 0 to 0.5mol/L of sodium chloride, has the pH value of 7.0 to 8.5 and has the concentration of 0.01 to 0.1 mol/L;

preferably, the filler for cation exchange chromatography is selected from fillers with filler ligands of sulfonic acid groups, phosphoric acid or phosphorous acid groups, phenolic hydroxyl groups and carboxyl groups; more preferably, the cationic filler is SP-Sepharose Fast Flow;

preferably, the dialyzed eluate is stored at 4 ℃;

4) carrying out affinity chromatography or composite filler chromatography on the eluent dialyzed in the step 3), carrying out linear gradient elution, collecting the eluent containing the active elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 5000-30000 kDa and dialysate;

wherein, the solid phase filler of the affinity chromatography is selected from affinity fillers containing two types of ligands; the composite filler for the composite filler chromatography is selected from hydroxyapatite;

wherein, in the linear gradient elution of the affinity chromatography, a gradient eluent A is an alkaline buffer solution containing 0-2mol/L NaCl, the pH value is 7.0-8.5, and the concentration is 0.01-0.1 mol/L; the gradient eluent B is an alkaline buffer solution with the concentration of 0.01-0.1mol/L and the pH value of 7.0-8.5, and contains 0-2mol/L sodium chloride and 0.05-0.5mol/L sugar competitive eluent;

in the linear gradient elution of the composite filler chromatography, a gradient eluent A is an alkaline buffer solution which contains 0-0.5mol/L NaCl, has a pH value of 7.0-8.5 and has a concentration of 0.01-0.1mol/L, and a gradient eluent B is an alkaline buffer solution which contains 0.6-2mol/L NaCl, has a pH value of 7.0-8.5 and has a concentration of 0.01-0.1 mol/L.

Wherein the dialysate is an alkaline buffer solution containing 0-0.1mol/L NaCl, pH7.0-8.9, and concentration 0.01-0.1mol/L,

preferably, the dialyzed eluate is stored at 4 ℃;

5) carrying out anion exchange chromatography and linear gradient elution on the eluent dialyzed in the step 4), collecting the eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 5000-30000 kDa and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an alkaline buffer solution which contains 0 to 0.1mol/L NaCl, has the pH value of 7.0 to 8.9 and has the concentration of 0.01 to 0.1 mol/L; the gradient eluent B is an alkaline buffer solution which contains 0.1-0.6mol/L NaCl, has the pH value of 7.0-8.9 and has the concentration of 0.01-0.1 mol/L;

wherein the dialysate is an alkaline buffer solution containing 0-0.1mol/L NaCl, with pH of 7.0-8.5 and concentration of 0.01-0.1 mol/L;

preferably, the anion exchange chromatography packing is selected from packing in which the packing ligands are of the quaternary ammonium, tertiary amine, secondary amine and primary amine group type; more preferably, the anion exchange chromatography packing is selected from DEAE-Sepharose Fast Flow or Capto DEAE;

preferably, the dialyzed eluate is stored at 4 ℃;

6) performing gel filtration chromatography on the dialyzed eluent, performing linear elution, and collecting the eluent containing the activity elution peak of the blood coagulation factor X activator to obtain the blood coagulation factor X activator;

preferably, the gel is Superdex G75;

wherein the eluent is an alkaline buffer solution containing 0-0.1mol/L NaCl, the pH value is 7.0-8.5, and the concentration is 0.01-0.1 mol/L.

Preferably, said collecting of the eluate containing the elution peak of the factor x activator activity is effected by a method comprising the steps of:

collecting eluate, performing ultraviolet spectrophotometer detection on the eluate at 280nm wavelength, collecting each elution peak according to ultraviolet absorption spectrum and determining activity, performing SDS-PAGE gel electrophoresis tracking detection, and collecting eluate containing active elution peak of blood coagulation factor X activator; more preferably, the activity assay uses the S-2765 substrate chromogenic method (refer to patent application No. CN 201510940717.9);

preferably, in the step 1), the pH value of the alkaline buffer solution is 7.5-8.5, and the concentration is 0.04-0.06 mol/L; further preferably, the pH of the alkaline buffer is 8.0, and the concentration is 0.05 mol/L;

preferably, in step 1), the centrifugation conditions are: at 4-8 deg.C, relative centrifugal force of 1200-20000 g, preferably 3000-4000 g, and centrifugation time of 5-20min, preferably 15 min;

preferably, in step 2), the gradient eluent A is an alkaline buffer solution with pH of 7.4-8.5 and concentration of 0.04-0.06 mol/L; more preferably, the gradient eluent A is an alkaline buffer solution with the pH of 8.0 and the concentration of 0.05 mol/L;

the gradient eluent B is an alkaline buffer solution which contains 0.4-0.6mol/L NaCl, has the pH value of 7.4-8.5 and has the concentration of 0.04-0.06 mol/L; more preferably, the gradient eluent B is an alkaline buffer solution containing 0.5mol/L NaCl and having a pH of 8.0 and a concentration of 0.05 mol/L;

preferably, in step 2), before anion exchange chromatography, 10 column volumes and more are equilibrated using an alkaline buffer solution having a pH of 7.0 to 8.9 and a concentration of 0.01 to 0.1mol/L as an equilibration solution; preferably, the equilibrium solution is an alkaline buffer solution with the pH of 7.4-8.5 and the concentration of 0.04-0.06 mol/L; more preferably, the equilibrium solution is an alkaline buffer solution with pH of 8.0 and concentration of 0.05 mol/L;

preferably, in step 2), the linear flow rate at equilibrium is between 60 and 200cm/h, more preferably 100 cm/h;

preferably, in step 2), the linear flow rate at elution is from 20 to 100cm/h, more preferably 60 cm/h;

preferably, in step 2), the dialysate is an acidic buffer solution with pH of 5.5-6.5 and concentration of 0.04-0.08 mol/L; further preferably, the dialysate is an acidic buffer solution with pH of 6.0 and concentration of 0.05 mol/L;

preferably, in step 3), the gradient eluent A is an acidic buffer solution with pH of 5.5-6.5 and concentration of 0.04-0.08 mol/L; further preferably, the gradient eluent A is an acidic buffer solution with pH of 6.0 and concentration of 0.05 mol/L;

more preferably, the gradient eluent B is an acidic buffer solution which contains 0.4-0.6mol/L NaCl, has the pH value of 5.5-6.5 and has the concentration of 0.04-0.08 mol/L; further preferably, the gradient eluent B is an acidic buffer solution containing 0.5mol/L NaCl and having a pH of 6.0 and a concentration of 0.05 mol/L;

preferably, in step 3), before the cation exchange chromatography, 10 column volumes and more are equilibrated using an acidic buffer solution having a pH of 5.0 to 6.8 and a concentration of 0.02 to 0.1mol/L as an equilibration solution; preferably, the equilibrium solution is an acidic buffer solution with pH of 5.5-6.5 and concentration of 0.04-0.08 mol/L; further preferably, the equilibrium solution is an acidic buffer solution with pH of 6.0 and concentration of 0.05 mol/L;

preferably, in step 3), the linear flow rate at equilibrium is between 60 and 200cm/h, more preferably 100 cm/h;

preferably, in step 3), the linear flow rate at elution is from 20 to 100cm/h, more preferably 60 cm/h;

preferably, in step 3), the affinity chromatography dialysate is a dialysate containing 0.2-1mol/L NaCl, 0.001-0.003mol/L CaCl₂And MnCl₂An alkaline buffer solution with pH of 7.6-8.4 and concentration of 0.02-0.05 mol/L; further preferably, the dialysate is a dialysate containing 0.8mol NaCl/L, 0.002mol/LCaCl₂And MnCl₂An alkaline buffer solution with pH of 8.0 and concentration of 0.03 mol/L;

preferably, in the step 3), the composite filler chromatography dialysate is an alkaline buffer solution which contains 0.2-0.4mol/L sodium chloride, has pH of 7.6-8.4 and has concentration of 0.02-0.05 mol/L; further preferably, the dialysate is an alkaline buffer solution containing 0.3mol/L sodium chloride, having a pH of 8.0 and a concentration of 0.03 mol/L;

preferably, in step 4), the affinity layer filler ligands are of the hyacinth bean lectin and concanavalin classes, respectively; more preferably Lentin Lectin Sepharose 4B, ConA Sepharose 4B or 6B, and still more preferably Lentin Lectin Sepharose 4B and ConA Sepharose 4B.

Preferably, in step 4), the hydroxyapatite composite-based filler is selected from a hydroxyfluorapatite, a hydroxyapatite and a fluoroapatite filler; more preferably CHT ceramic hydroxyapatite I;

preferably, in the step 4), the gradient eluent A is an alkaline buffer solution containing 0.2-1mol/L NaCl, pH 7.6-8.4 and concentration 0.02-0.05mol/L in the affinity chromatography; further preferably, the gradient eluent A is an alkaline buffer solution containing 0.8mol NaCl/L, pH of 8.0 and having a concentration of 0.03 mol/L; in the affinity chromatography, the gradient eluent B is an alkaline buffer solution which contains 0.1-1mol/L NaCl and 0.1-0.5mol/L saccharide competitive eluent and has the pH of 7.6-8.4 and the concentration of 0.02-0.05 mol/L; more preferably, the gradient eluent B is an alkaline buffer solution with a pH of 8.0 and a concentration of 0.03mol/L containing 0.8mol/L NaCl and 0.2-0.4mol/L saccharide competitive eluent;

preferably, in the step 4), in the chromatography of the composite filler, the gradient eluent A is an alkaline buffer solution which contains 0.2-0.4mol/L NaCl, has the pH value of 7.6-8.4 and has the concentration of 0.02-0.05 mol/L; more preferably, the gradient eluent A is an alkaline buffer solution containing 0.3mol/L NaCl and having a pH of 8.0 and a concentration of 0.03 mol/L;

in the chromatography of the composite filler, the gradient eluent B is an alkaline buffer solution which contains 0.6-1mol/L NaCl, has the pH value of 7.6-8.4 and has the concentration of 0.02-0.05 mol/L; more preferably, the gradient eluent B is an alkaline buffer solution containing 0.8mol/L NaCl, with the pH value of 8.0 and the concentration of 0.03 mol/L;

preferably, in step 4), the saccharide-type competitive eluent is selected from the group consisting of methylglucose-type competitive eluent and methylpyrano-saccharide-type competitive eluent;

preferably, in step 4), 0.001 to 0.005mol/LCaCl containing 0 to 2mol/LNaCl is used before performing the affinity chromatography₂And MnCl₂Alkaline buffer solution with pH of 7.0-8.5 and concentration of 0.01-0.1mol/L is used as equilibrium solution to balance 10 column volumes and more; preferably, the equilibrium liquid contains 0.2-1mol/L NaCl and 0.001-0.003mol/LCaCl₂And MnCl₂An alkaline buffer solution with pH of 7.6-8.4 and concentration of 0.02-0.05 mol/L; more preferably, the equilibrium solution is 0.8mol/L NaCl and 0.002mol/LCaCl₂And MnCl₂An alkaline buffer solution with pH of 8.0 and concentration of 0.03 mol/L;

preferably, in step 4), before the chromatography of the composite packing is performed, 10 column volumes and more are equilibrated with an alkaline buffer solution containing 0 to 0.5mol/L NaCl, pH7.0 to 8.5, and having a concentration of 0.01 to 0.1mol/L, as an equilibration solution; preferably, the equilibrium solution is an alkaline buffer solution which contains 0.2-0.4mol/L NaCl, has the pH value of 7.6-8.4 and has the concentration of 0.02-0.05 mol/L; further preferably, the equilibrium solution is an alkaline buffer solution containing 0.3mol/L NaCl, having a pH of 8.0 and a concentration of 0.03 mol/L;

preferably, in step 4), the linear flow rate at the time of affinity chromatography equilibration is 30-70cm/h, more preferably 60 cm/h;

preferably, in step 4), the linear flow rate at elution of affinity chromatography is from 30 to 70cm/h, more preferably 40 cm/h;

preferably, in the step 4), the linear flow rate of the composite filler at chromatographic equilibrium is 50-200cm/h, more preferably 100 cm/h;

preferably, in step 4), the linear flow rate of the composite chromatography elution is 50-100cm/h, more preferably 80 cm/h;

preferably, in step 4), the dialysate is an alkaline buffer solution containing 0.05-0.1mol/L NaCl, having a pH of 7.5-8.5, and having a concentration of 0.02-0.05 mol/L; further preferably, the dialysate is an alkaline buffer solution containing 0.1mol/L NaCl and having a pH of 8.0 and a concentration of 0.03 mol/L;

preferably, in step 5), the gradient eluent A is an alkaline buffer solution containing 0.05-0.1mol/L NaCl, with the pH value of 7.5-8.5 and the concentration of 0.02-0.05 mol/L; more preferably, the gradient eluent A is an alkaline buffer solution containing 0.1mol of NaCl and having a pH of 8.0 and a concentration of 0.03 mol/L;

the gradient eluent B is an alkaline buffer solution which contains 0.4-0.6mol/L NaCl, has the pH value of 7.5-8.5 and has the concentration of 0.02-0.05 mol/L; more preferably, the gradient eluent B is an alkaline buffer solution containing 0.5mol/L NaCl and having a pH of 8.0 and a concentration of 0.03 mol/L;

preferably, in step 5), before anion exchange chromatography, 10 column volumes and more are equilibrated using an alkaline buffer solution containing 0 to 0.1mol/L NaCl, having a pH of 7.0 to 8.9, and having a concentration of 0.01 to 0.1mol/L, as an equilibration solution; preferably, the equilibrium solution is an alkaline buffer solution with the pH value of 7.5-8.5 and the concentration of 0.02-0.05mol/L, wherein the pH value of the alkaline buffer solution contains 0.05-0.1mol/L NaCl; more preferably, the equilibrium solution is an alkaline buffer solution containing 0.1mol/L NaCl and having a pH of 8.0 and a concentration of 0.03 mol/L;

preferably, in step 5), the linear flow rate at equilibrium is between 60 and 200cm/h, more preferably 100 cm/h;

preferably, in step 5), the linear flow rate at elution is from 20 to 100cm/h, more preferably 60 cm/h;

preferably, in step 5), the dialysate is an alkaline buffer solution containing 0.01-0.05mol/L NaCl, having a pH of 7.5-8.5 and a concentration of 0.01-0.05 mol/L; further preferably, the dialysate is an alkaline buffer solution containing 0.05mol/L NaCl, having a pH of 8.0 and a concentration of 0.02 mol/L;

preferably, in step 6), the eluent is an alkaline buffer solution containing 0.01-0.05mol/L NaCl, with the pH value of 7.5-8.5 and the concentration of 0.01-0.05 mol/L; further preferably, the eluent is an alkaline buffer solution containing 0.05mol/L NaCl and having the pH value of 8.0 and the concentration of 0.02 mol/L;

preferably, in step 6), 5 column volumes or more are equilibrated with an alkaline buffer solution containing 0 to 0.1mol/L NaCl, having a pH of 7.0 to 8.5 and a concentration of 0.01 to 0.1mol/L, as an equilibration solution before performing gel filtration chromatography; preferably, the equilibrium solution is an alkaline buffer solution with 0.01-0.05mol/L of NaCl, pH7.5-8.5 and concentration of 0.01-0.05 mol/L; more preferably, the equilibrium solution is an alkaline buffer solution containing 0.05mol/L NaCl and having a pH of 8.0 and a concentration of 0.02 mol/L;

preferably, in step 6), the linear flow rate at equilibrium is from 20 to 80cm/h, more preferably 60 cm/h;

preferably, in step 6), the linear flow rate at elution is 20-60cm/h, more preferably 40 cm/h;

preferably, in step 6), the separation range of the gel packing is 3000-300000 Da.

Preferably, the alkaline buffer is selected from the group consisting of Tris-Hcl buffer, phosphate buffer and phosphate-citrate buffer;

preferably, the acidic buffer is selected from the group consisting of phosphate buffer, phosphate-citrate buffer and acetate buffer;

in another aspect, the invention provides the use of a factor X activator of the invention in the manufacture of a medicament for the treatment of a bleeding disorder;

preferably, the bleeding includes clinical bleeding, surgical bleeding, and various surgical acute bleeding.

In yet another aspect, the present invention provides a pharmaceutical composition for treating bleeding, comprising the factor X activator and a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the following advantages:

1) the coagulation factor X activator provided by the invention has high purity, the purity is 100% by using a size exclusion chromatography SEC column for detection, and the purity is up to 96% by using a C4 reverse phase column for detection.

2) The method provided by the invention firstly uses anion-cation exchange filler chromatography and then carries out affinity chromatography, so that the purity of the blood coagulation factor X activator after the affinity chromatography is close to that of electrophoresis, and the service life of the affinity filler is greatly prolonged, thereby saving the production cost and being beneficial to large-scale industrial production.

As known to those skilled in the art, most of the ligands of the affinity packing materials at present are easily exfoliated during complicated chromatography of sample components, and the price of the packing materials is high, so in practical applications, affinity chromatography is usually used in a fine separation or a fine separation step after crude protein separation. The inventor of the invention finds that when the activator of the blood coagulation factor X is prepared, the first two steps adopt anion-cation exchange packing, most of active components without the activator of the blood coagulation factor X can be removed, and a purer sample is provided for the next step of affinity chromatography, so that the nonspecific adsorption of impurity protein in the process of the affinity chromatography is greatly reduced, the loading capacity of the packing on the activator of the blood coagulation factor X is improved, and the purity of the activator of the blood coagulation factor X after the affinity column chromatography is close to that of electrophoresis.

3) The inventor finds that the specific affinity filler can be specifically combined with the activator of the blood coagulation factor X in the venom of the Bothrops atrox, thereby greatly improving the production efficiency.

4) The preparation method of the blood coagulation factor X activator is particularly suitable for producing a human medicine mode, a chromatographic column used by the method can be amplified according to the same proportion of preparation requirements, the process is stable, and the chromatographic process is controllable;

5) the method of the invention adopts a membrane package or a membrane stack device in the dialysis process, has short dialysis time (completed within 2 h), is convenient, can keep the sterility in the dialysis process, and can remove small molecular impurities such as polypeptide and the like in the snake venom solution in the dialysis process.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a high performance liquid SEC column purity assay of a factor X activator of the present invention;

FIG. 2 is a high performance liquid phase reverse phase C4 column purity assay for a factor X activator of the present invention;

FIG. 3 shows the factor X activator SDS-PAGE purity of the invention (example one);

FIG. 4 shows the factor X activator SDS-PAGE purity of the invention (example two);

FIG. 5 shows the results of MALDI-TOF molecular weight determination of a factor X activator of the present invention;

FIG. 6 is a MALDI-TOF molecular weight mass spectrum of a coagulation factor X activator of the present invention

Detailed Description

The present invention will be further illustrated by the following examples, but the present invention is not limited to these examples, which are provided for more detailed explanation and should not be construed as limiting the present invention in any way. Certain changes and modifications within the scope of the claims by one skilled in the art are also to be considered within the scope of the invention.

Example 1 isolation and purification of Agkistrodon halys venom factor X activator

1. Snake venom solubilization

Dissolving 10g of Bothrops atrox venom (venom from biological pharmacy Co., Ltd., Tao-Ning-Daknookang) in 100ml of Tris-HCl buffer solution (pH 8.0, 0.05 mol/L) at 4-8 deg.C, centrifuging for 15min at 3000g, and collecting supernatant.

2. DEAE-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

2-1) DEAE-Sepharose Fast Flow column packing: about 90ml of DEAE-Sepharose Fast Flow filler preserved in 20% ethanol is taken, purified water is firstly used for replacement and alcohol removal, a 2.6X 20cm chromatographic column is filled, then Tris-HCl buffer solution with the pH value of 8.0 and the concentration of 0.05mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

2-2) loading and eluting: pumping the venom supernatant centrifuged in the step 2-1) into a balanced chromatographic column at a linear flow rate of 60cm/h, after the sample loading is finished, carrying out balanced elution by using Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L, a gradient eluent B is 300ml of Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L and containing 0.5mol/L sodium chloride, the linear flow rate is 60cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 8 chromatographic peaks in total. The activity determination and gel electrophoresis tracing confirm that the 6 th peak is the main peak of the activity of the factor X activator.

2-3) dialysis of the sample: and (3) dialyzing the active peak collected in the step 2-2) against 0.05mol/L phosphate buffer solution with pH of 6.0 by using a membrane (purchased from materials of Saedodes and polyether sulfone) with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

3. SP-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

3-1) SP-Sepharose Fast Flow column packing: about 90ml of SP-Sepharose Fast Flow filler stored in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a 2.6X 20cm chromatographic column is filled, then more than 10 column volumes are balanced by phosphate buffer solution with pH6.0 and 0.05mol/L, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

3-2) loading and eluting: pumping the snake venom solution dialyzed in the step 2 into a well balanced chromatographic column, carrying out linear flow velocity of 60cm/h, carrying out balanced elution by using phosphate buffer solution with pH of 6.0 and 0.05mol/L after the sample loading is finished, carrying out linear gradient elution after the ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of phosphate buffer solution with pH of 6.0 and 0.05mol/L, a gradient eluent B is 300ml of phosphate buffer solution with pH of 6.0 and 0.05mol/L and containing 0.5mol/L sodium chloride, the linear flow velocity is 60cm/h, collecting each peak according to an ultraviolet detection map with the wavelength of 280nm, and collecting 4 chromatographic peaks in total. And determining the 3 rd peak as the main active peak of the X factor activator by activity measurement and gel electrophoresis tracking.

3-3) dialysis of the sample: and 3-2) dialyzing the Tris-HCl buffer solution containing 0.8mol/L sodium chloride, 0.002mol/L calcium chloride and 0.002mol/L manganese chloride and having the pH value of 8.0 and 0.03mol/L by using a membrane with the cut-off molecular weight of 10000Da for the active peak collected in the step, and fixing the volume of the sample to about 80 ml. Placing in a refrigerator at 4 ℃ for standby.

4. Lentin Lectin Sepharose 4B column chromatography 1.6X 20cm (Filler from GE)

4-1) Lentil Lectin Sepharose 4B column: taking about 40ml of Lentil Lectin Sepharose 4B filler stored in 20% ethanol, firstly replacing with purified water to remove alcohol, filling a chromatographic column of 1.6 multiplied by 20cm, and then balancing more than 10 column volumes by Tris-HCl buffer solution containing 0.8mol/L sodium chloride, 0.002mol/L calcium chloride and 0.002mol/L manganese chloride, wherein the pH value is 8.0 and the pH value is 0.03mol/L, the linear flow rate is 60cm/h, and the ultraviolet absorption detection is carried out at the wavelength of 280 nm.

4-2) loading and eluting: pumping the dialyzed snake venom solution obtained in the step 3 into a balanced chromatographic column, carrying out linear flow rate of 40cm/h, carrying out balanced elution by using Tris-HCl buffer solution with the pH value of 8.0 and 0.03mol/L, wherein the Tris-HCl buffer solution contains 0.8mol/L sodium chloride, 0.002mol/L calcium chloride and 0.002mol/L manganese chloride, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is Tris-HCl buffer solution with the pH value of 8.0 and 0.03mol/L, the gradient eluent B is methyl-alpha-D-glucopyranoside with the pH value of 8.0 and the Tris-HCl buffer solution with the pH value of 0.03mol/L, carrying out linear flow rate of 40cm/h, collecting peaks according to a 280nm wavelength ultraviolet detection map, and collecting 3 chromatographic peaks in total. And determining that the 2 nd peak is the main activity peak of the X factor activator by activity measurement and gel electrophoresis tracking.

4-3) dialysis of the sample: and (3) dialyzing the active peak collected in the step 4-2) against Tris-HCl buffer solution containing 0.1mol/L sodium chloride, pH8.0 and 0.03mol/L by using a membrane with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

5. DEAE-Sepharose Fast Flow column chromatography 1.6X 20cm (Filler from GE)

5-1) DEAE-Sepharose Fast Flow column packing: about 40ml of DEAE-Sepharose Fast Flow filler preserved in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a chromatographic column of 1.6 multiplied by 20cm is filled, and then Tris-HCl buffer solution containing 0.1mol/L sodium chloride, pH8.0 and 0.03mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection is carried out at the wavelength of 280 nm.

5-2) loading and eluting: pumping the snake venom solution dialyzed in the step 4 into a balanced chromatographic column, carrying out linear flow rate of 60cm/h for 60cm/h, carrying out balanced elution by using Tris-HCl buffer solution containing 0.1mol/L of sodium chloride, pH8.0 and 0.03mol/L after the sample loading is finished, carrying out linear gradient elution after ultraviolet absorption reaches a base line, wherein a gradient eluent A is 150ml of Tris-HCl buffer solution containing 0.1mol/L of sodium chloride, pH8.0 and 0.03mol/L, a gradient eluent B is 150ml of Tris-HCl buffer solution containing 0.5mol/L of sodium chloride, pH8.0 and 0.03mol/L, the linear flow rate is 60cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 3 chromatographic peaks in total. And determining that the 2 nd peak is the main peak of the activity of the factor X activator by activity measurement and gel electrophoresis tracking.

5-3) sample dialysis: and (3) dialyzing and concentrating the activity peak collected in the step 5-2) by using a membrane with the cut-off molecular weight of 10000Da on Tris-HCl buffer solution containing 0.05mol/L sodium chloride, pH8.0 and 0.02mol/L, and fixing the volume of the sample to about 50 ml. Placing in a refrigerator at 4 ℃ for standby.

6. Superdex G75 column chromatography 2.6X 70cm (Filler from GE)

6-1) Superdex G75 column packing: about 350ml of Superdex G75 filler preserved in 20% ethanol is taken, purified water is firstly used for replacement and alcohol removal, then a 2.6X 70cm chromatographic column is filled, and then a Tris-HCl buffer solution containing 0.05mol/L sodium chloride, pH8.0 and 0.02mol/L is used for balancing more than 5 column volumes, the linear flow rate is 60cm/h, and the ultraviolet absorption with the wavelength of 280nm is used for detection.

6-2) loading and eluting: and (3) absorbing 10ml of sample liquid from the step 5 by using a syringe, loading the sample liquid, slowly adding the sample liquid along the side wall of the column in a rotating manner, after the sample liquid completely enters the filler, using less than 0.2ml of Tris-HCl buffer solution containing 0.05mol/L sodium chloride, having pH of 8.0 and 0.02mol/L to rinse the side wall and the gel surface, adding 10ml of buffer solution, and eluting at the linear flow rate of 40 cm/h. Collecting each peak according to an ultraviolet spectral width detection spectrum under 280nm, and collecting 1 chromatographic peak in total.

7. And (4) analyzing results:

7-1) purity detection

The high performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, SEC-HPLC: G2000 WXL; RP-HPLC: C4 chromatographic column) and an electrophoresis system are adopted for detection, the SEC-HPLC detection shows a single peak (shown in figure 1), the RP-HPLC detection shows a single peak (shown in figure 2), and the contents of the coagulation factor X activator are respectively 100% and 96.2% according to the area normalization method. SDS-PAGE denaturing non-reducing electrophoresis detects the target protein component showing a single band (as shown in FIG. 3).

7-2) yield and specific activity

The activity detection of the target protein is carried out by an S2765 substrate method, the total activity of the coagulation factor X activator in 10g of dissolved solution is 60 ten thousand units, the activity collected after chromatographic purification is 12 ten thousand units, and the yield is 20%. The specific activity was 4491U/mg.

7-3) structural characteristics of Agkistrodon halys factor X activator

MALDI-TOF Mass Spectrometry the molecular weight of the factor X activator was 87540Da, as shown in FIGS. 5 and 6.

B. Solid phase pH gradient (IPG) isoelectric focusing method (Bio-Rad manual) to obtain isoelectric point of 4.94-6.64;

C. reduced SDS-PAGE and Edman sequencing (sequencer: ABI Procise 491):

the reduced SDS-PAGE presents three bands, and the measured molecular weights are respectively H: 54 kDa; l1: 12 kDa; l2:13 kDa.

H chain N-terminal sequence SEQ ID NO: 1: LTPEQQAYLDAKKYV, respectively;

chain N-terminal sequence of L1: 2, SEQ ID NO: DCPSDWSSYEGHCYR, respectively;

chain N-terminal sequence of L2: 3, SEQ ID NO: DCNSYWSTYEGRSYR are provided.

D. The carbohydrase cleavage showed a sugar site in the heavy chain, being the N-sugar.

Example 2 isolation and purification of Agkistrodon halys venom factor X activator

1. Snake venom solubilization

Dissolving 10g of Bothrops atrox venom (purchased from biological pharmacy Co., Ltd., Tao-Ning-Daknookang) in 100ml of Tris-HCl buffer solution (pH 7.4) with concentration of 0.01mol/L at 4-8 deg.C, centrifuging for 15min at 3000g, and collecting supernatant.

2. DEAE-Sepharose Fast Flow column chromatography: 2.6X 20cm (from GE)

2-1) DEAE-Sepharose Fast Flow column packing: about 90ml of DEAE-Sepharose Fast Flow filler preserved in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a 2.6X 20cm chromatographic column is filled, then Tris-HCl buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

2-2) loading and eluting: pumping the centrifuged snake venom supernatant into a balanced chromatographic column, carrying out linear flow rate of 60cm/h, carrying out balanced elution by using Tris-HCl buffer solution with the pH value of 7.4 and 0.01mol/L after the sample loading is finished, carrying out linear gradient elution after the ultraviolet absorption is reduced to a baseline, carrying out linear gradient elution by using 300ml of Tris-HCl buffer solution with the pH value of 7.4 and 0.01mol/L as a gradient eluent A, 300ml of Tris-HCl buffer solution with the pH value of 7.4 and 0.01mol/L as a gradient eluent B and carrying out linear flow rate of 60cm/h, collecting each peak according to an ultraviolet detection map with the wavelength of 280nm, and collecting 8 chromatographic peaks in total. And the activity measurement and gel electrophoresis tracking determine that the 6 th peak is the main activity peak of the factor X activator.

2-3) dialysis of the sample: and (3) dialyzing the active peak collected in the step 2-2) against 0.1mol/L sodium acetate buffer solution with pH of 5.0 by using a membrane (purchased from materials of Saedodes and polyether sulfone) with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

3. SP-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

3-1) SP-Sepharose Fast Flow column packing: about 90ml of SP-Sepharose Fast Flow filler stored in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a 2.6X 20cm chromatographic column is filled, then more than 10 column volumes are balanced by sodium acetate buffer solution with pH5.0 and 0.1mol/L, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

3-2) loading and eluting: pumping the snake venom solution dialyzed in the step 2 into a well balanced chromatographic column, carrying out linear flow velocity of 60cm/h, carrying out balanced elution by using an acetic acid buffer solution with the pH value of 5.0 and 0.1mol/L after the sample loading is finished, carrying out linear gradient elution after the ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of a sodium acetate buffer solution with the pH value of 5.0 and 0.1mol/L, a gradient eluent B is 300ml of a sodium acetate buffer solution with the pH value of 5.0 and 0.1mol/L and containing 0.6mol/L sodium chloride, the linear flow velocity of 60cm/h, collecting each peak according to an ultraviolet detection map with the wavelength of 280nm, and collecting 4 chromatographic peaks in total. The activity determination and gel electrophoresis tracing confirm that the 3 rd peak is the main peak of the activity of the factor X activator.

3-3) dialysis of the sample: and 3-2) dialyzing the active peak collected in the step against 0.3mol/L sodium chloride-containing phosphate buffer solution (pH8.0 and 0.03 mol/L) by using a membrane (purchased from Sidolide, a polyether sulfone material) with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 80 ml. Placing in a refrigerator at 4 ℃ for standby.

4. CHT ceramic hydroxyapatite I column chromatography 1.6X 20cm (from Bole Co.)

4-1) packing CHT ceramic hydroxyapatite I into a column: soaking CHT ceramic hydroxyapatite I dry powder in 0.3mol/L NaCl-containing phosphate buffer solution with pH of 8.0 and 0.03mol/L for 4h, filling about 40ml of filler into a chromatographic column of 1.6X 20cm, and balancing 10 column volumes by 0.3mol/L NaCl-containing phosphate buffer solution with pH of 8.0 and 0.03mol/L, wherein the linear flow rate is 100cm/h, and ultraviolet absorption detection is carried out at the wavelength of 280 nm.

4-2) loading and eluting: pumping the snake venom solution dialyzed in the step 3 into a balanced chromatographic column, carrying out balanced elution at a linear flow rate of 80cm/h, after the sample loading is finished, carrying out balanced elution by using a phosphate buffer solution containing 0.3mol/L NaCl and having a pH value of 8.0 and 0.03mol/L, carrying out linear gradient elution after ultraviolet absorption reaches a baseline, wherein a gradient eluent A is 150ml of a phosphate buffer solution containing 0.3mol/L sodium chloride and having a pH value of 8.0 and 0.03mol/L, a gradient eluent B is 150ml of a phosphate buffer solution containing 0.8mol/L sodium chloride and having a pH value of 8.0 and 0.03mol/L, the linear flow rate is 80cm/h, collecting each peak according to an ultraviolet spectral width detection map under 280nm, and collecting 3 chromatographic peaks in total. And determining that the 2 nd peak is the main peak of the activity of the factor X activator by activity measurement and gel electrophoresis tracking.

4-3) dialysis of the sample: and (3) dialyzing the active peak collected in the step 4-2) against Tris-HCl buffer solution containing 0.05mol/L sodium chloride, pH7.4 and 0.08mol/L by using a membrane (purchased from Sidolide, a polyether sulfone material) with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

5. DEAE-Sepharose Fast Flow column chromatography 1.6X 20cm (Filler from GE)

5-1) DEAE-Sepharose Fast Flow column packing: about 40ml of DEAE-Sepharose Fast Flow filler preserved in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a chromatographic column of 1.6 multiplied by 20cm is filled, and then Tris-HCl buffer solution containing 0.05mol/L sodium chloride, pH7.4 and 0.08mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection is carried out at the wavelength of 280 nm.

5-2) loading and eluting: pumping the snake venom solution dialyzed in the step 4 into a balanced chromatographic column, carrying out equilibrium elution at a linear flow rate of 80cm/h, after the sample loading is finished, using Tris-HCl buffer solution containing 0.05mol/L of sodium chloride, pH7.4 and 0.08mol/L to carry out equilibrium elution, carrying out linear gradient elution after ultraviolet absorption reaches a base line, wherein a gradient eluent A is 150ml of Tris-HCl buffer solution containing 0.05mol/L of sodium chloride, pH7.4 and 0.08mol/L, a gradient eluent B is 150ml of Tris-HCl buffer solution containing 0.5mol/L of sodium chloride, pH7.4 and 0.08mol/L, the linear flow rate is 80cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 2 chromatographic peaks in total. And determining that the 2 nd peak is the main peak of the activity of the factor X activator by activity measurement and gel electrophoresis tracking.

5-3) sample dialysis: and (3) dialyzing and concentrating the active peak collected in the step 5-2) by using a membrane package (purchased from Sidolide and polyether sulfone materials) with the cut-off molecular weight of 10000Da on Tris-HCl buffer solution containing 0.1mol/L of sodium chloride, pH7.5 and 0.05mol/L, and fixing the volume of the sample to about 50 ml. Placing in a refrigerator at 4 ℃ for standby.

6. Superdex G75 column chromatography 2.6X 70cm (Filler from GE)

6-1) Superdex G75 column packing: about 350ml of Superdex G75 filler preserved in 20% ethanol is taken, purified water is firstly used for replacement and alcohol removal, then a 2.6X 70cm chromatographic column is filled, and then Tris-HCl buffer solution containing 0.1mol/L sodium chloride, pH7.5 and 0.05mol/L is used for balancing more than 5 column volumes, the linear flow rate is 50cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

6-2) loading and eluting: and (3) absorbing 10ml of sample liquid from the step (5) by using a syringe, loading the sample liquid, slowly adding the sample liquid along the side wall of the column in a rotating manner, after the sample liquid completely enters the filler, using less than 0.2ml of Tris-HCl buffer solution containing 0.1mol/L sodium chloride and having pH of 7.5 and 0.05mol/L to rinse the side wall and the gel surface, adding 10ml of buffer solution, and eluting at the linear flow rate of 40 cm/h. Collecting each peak according to a 280nm wavelength ultraviolet detection spectrum, and collecting 1 chromatography peak in total.

7. And (4) analyzing results:

7-1) purity detection

High performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, SEC-HPLC: G2000 WXL; RP-HPLC: C4 chromatographic column) and an electrophoresis system are adopted for detection, the SEC-HPLC detection shows a single peak, the RP-HPLC detection shows a single peak, and the contents of the blood coagulation factor X activator are respectively 100% and 95.7% according to the area normalization method. SDS-PAGE denaturing non-reducing electrophoresis detection of the target protein fraction revealed a single band (as shown in FIG. 4).

7-2) yield and specific activity

The activity detection of the target protein is carried out by an S2765 substrate method, the total activity of the coagulation factor X activator in 10g of dissolved solution is 65 ten thousand units, the activity collected after chromatographic purification is 11.5 ten thousand units, and the yield is 17.7%. The specific activity was 4282U/mg.

Example 3 spearhead Agkistrodon halys snakeIsolation and purification of toxic factor X activator

1. Dissolving snake venom:

dissolving 10g Bothrops atrox venom with 100ml Tris-HCl buffer solution (pH 8.5 and 0.1 mol/L) at 4-8 deg.C, centrifuging 3000g for 15min, and collecting supernatant.

2. DEAE-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

2-1) DEAE-Sepharose Fast Flow column packing: about 90ml of DEAE-Sepharose Fast Flow filler preserved in 20% ethanol is taken, purified water is firstly used for replacement and alcohol removal, then a 2.6X 20cm chromatographic column is filled, and then Tris-HCl buffer solution with the pH value of 8.5 and the concentration of 0.1mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

2-2) loading and eluting: pumping the venom supernatant centrifuged in the step 2-1) into a balanced chromatographic column at a linear flow rate of 60cm/h, after the sample loading is finished, carrying out balanced elution by using a Tris-HCl buffer solution with the pH value of 8.5 and 0.1mol/L, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of a Tris-HCl buffer solution with the pH value of 8.5 and 0.1mol/L, a gradient eluent B is 300ml of a Tris-HCl buffer solution with the pH value of 8.5 and 0.1mol/L and containing 0.5mol/L sodium chloride, the linear flow rate is 60cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 7 chromatographic peaks in total. And the activity measurement and gel electrophoresis tracking determine that the 6 th peak is the main activity peak of the factor X activator.

2-3) dialysis of the sample: and (3) dialyzing the phosphate buffer solution with pH6.8 and 0.02mol/L by using a membrane package with cut-off molecular weight of 10000Da for the active peak collected in the step 2-2), and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

3. SP-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

3-1) SP-Sepharose Fast Flow column packing: about 90ml of SP-Sepharose Fast Flow filler stored in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a 2.6X 20cm chromatographic column is filled, then more than 10 column volumes are balanced by phosphate buffer solution with pH6.8 and 0.02mol/L, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

3-2) loading and eluting: pumping the snake venom solution dialyzed in the step 2 into a well balanced chromatographic column, carrying out linear flow velocity of 60cm/h, carrying out balanced elution by using phosphate buffer solution with pH of 6.8 and 0.02mol/L after the sample loading is finished, carrying out linear gradient elution after the ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of phosphate buffer solution with pH of 6.8 and 0.02mol/L, a gradient eluent B is 300ml of phosphate buffer solution with pH of 6.8 and 0.02mol/L and containing 0.5mol/L sodium chloride, the linear flow velocity is 60cm/h, collecting each peak according to an ultraviolet detection map with the wavelength of 280nm, and collecting 4 chromatographic peaks in total. The activity determination and gel electrophoresis tracing confirm that the 3 rd peak is the main peak of the activity of the factor X activator.

3-3) dialysis of the sample: the active peak collected in the step 3-2) is subjected to membrane treatment by using a membrane with the cut-off molecular weight of 10000Da and contains 0.3mol/L NaCl and 0.005mol/L LCaCl₂And MnCl₂And a phosphate buffer solution with pH of 7.5 and a concentration of 0.05mol/L is dialyzed, and the volume of the sample is determined to be about 80 ml. Placing in a refrigerator at 4 ℃ for standby.

4. ConA Sepharose 4B column chromatography 1.6X 20cm (Filler from GE)

4-1) ConA Sepharose 4B column packing: about 40ml of ConA Sepharose 4B filler stored in 20% ethanol was taken, the ethanol was removed by substitution with purified water, and the column was packed in a 1.6X 20cm column, followed by column chromatography using a column containing 0.3mol/L NaCl and 0.005mol/L NaCl₂And MnCl₂The pH value is 7.5, the concentration is 0.05mol/L phosphate buffer solution is balanced for more than 10 column volumes, the linear flow rate is 60cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

4-2) loading and eluting: pumping the snake venom solution dialyzed in the step 3 into a well balanced chromatographic column, carrying out linear flow velocity of 40cm/h, carrying out balanced elution by using phosphate buffer solution with pH7.5 and 0.05mol/L after loading, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, carrying out gradient elution by using a gradient eluent A of 150ml of phosphate buffer solution with pH7.5 and 0.05mol/L, using a gradient eluent B of 150ml of phosphate buffer solution with pH7.5 and 0.05mol/L containing 1mol/L sodium chloride, carrying out linear flow velocity of 40cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 3 chromatographic peaks in total. And determining that the 2 nd peak is the main activity peak of the X factor activator by activity measurement and gel electrophoresis tracking.

4-3) dialysis of the sample: and (3) dialyzing the active peak collected in the step 4-2) against Tris-HCl buffer solution containing 0.1mol/L sodium chloride, pH8.5 and 0.1mol/L by using a membrane with the cut-off molecular weight of 10000Da, and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

5. DEAE-Sepharose Fast Flow column chromatography 1.6X 20cm (Filler from GE)

5-3) DEAE-Sepharose Fast Flow column packing: about 40ml of DEAE-Sepharose Fast Flow filler stored in 20% ethanol is taken, purified water is used for replacement and alcohol removal, a chromatographic column of 1.6 multiplied by 20cm is filled, and then Tris-HCl buffer solution containing 0.1mol/L sodium chloride, pH8.5 and 0.1mol/L is used for balancing more than 10 column volumes, the linear Flow rate is 100cm/h, and the ultraviolet absorption detection of 280nm wavelength is carried out.

5-2) loading and eluting: pumping the snake venom solution dialyzed in the step 4 into a well balanced chromatographic column, carrying out linear flow velocity of 60cm/h, after the sample loading is finished, carrying out balanced elution by using Tris-HCl buffer solution containing 0.1mol/L of sodium chloride, pH8.5 and 0.1mol/L, carrying out linear gradient elution after ultraviolet absorption is reduced to a base line, wherein a gradient eluent A is 150ml of Tris-HCl buffer solution containing 0.1mol/L of sodium chloride, pH8.5 and 0.0mol/L, a gradient eluent B is 150ml of Tris-HCl buffer solution containing 0.5mol/L of sodium chloride, pH8.5 and 0.1mol/L, the linear flow velocity of 60cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 2 chromatographic peaks in total. And determining the 1 st peak as the main activity peak of the X factor activator by activity measurement and gel electrophoresis tracking.

5-3) sample dialysis: and (3) dialyzing and concentrating the active peak collected in the step 5-2) by using a membrane package with the cut-off molecular weight of 10000Da on phosphate buffer solution with the pH value of 8.0 and the concentration of 0.05mol/L, and fixing the volume of the sample to about 50 ml. Placing in a refrigerator at 4 ℃ for standby.

6. Superdex G75 column chromatography 2.6X 70cm (Filler from GE)

6-1) Superdex G75 column packing: about 350ml of Superdex G75 filler preserved in 20% ethanol is taken, purified water is firstly used for replacement and alcohol removal, then a 2.6X 70cm chromatographic column is filled, and then more than 5 column volumes are balanced by phosphate buffer with pH8.0 and 0.05mol/L, the linear flow rate is 50cm/h, and the ultraviolet absorption detection with the wavelength of 280nm is carried out.

6-2) loading and eluting: and (3) absorbing 10ml of sample liquid from the step (5) by using a syringe, loading the sample liquid, slowly adding the sample liquid along the side wall of the column in a rotating manner, after the sample liquid completely enters the filler, using less than 0.2ml of phosphate buffer solution with the pH value of 8.0 and the concentration of 0.05mol/L to rinse the side wall and the rubber surface, adding 10ml of buffer solution, and then eluting at the linear flow rate of 40 cm/h. Collecting each peak according to an ultraviolet spectral width detection spectrum under 280nm, and collecting 1 chromatographic peak in total.

7. And (4) analyzing results:

7-1) purity detection

High performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, SEC-HPLC: G2000 WXL; RP-HPLC: C4 chromatographic column) and an electrophoresis system are adopted for detection, the SEC-HPLC detection shows a single peak, the RP-HPLC detection shows a single peak, and the contents of the blood coagulation factor X activator are respectively 100% and 94.7% according to the area normalization method. SDS-PAGE denaturing non-reducing electrophoresis detection of the target protein component shows a single band.

7-2) yield and specific activity

The activity detection of the target protein is carried out by an S2765 substrate method, the total activity of the coagulation factor X activator in 10g of dissolved solution is 63 ten thousand units, the activity collected after chromatographic purification is 12.2 ten thousand units, and the yield is 19.4%. The specific activity is 4260U/mg.

Example 4 Studies on the function of Agkistrodon halys venom activator of factor X

This study examined the procoagulant effects of boagkistrodon spearhead activator of coagulation factor X.

Experiment one

1. Purpose of experiment

The effect of a factor X activator of Agkistrodon halys on partial thrombin activation time (aPTT) in human normal plasma and factor VIII and factor IX-poor plasma was examined.

2. Reagent and instrument

Normal coagulation quality control plasma German Mei Chun company batch No. 047B-D087A

Factor VIII-poor plasma Siemens Ltd, batch No. 503627

Factor IX deficient plasma siemens company batch number 500874D

aPTT kit German Mei Chun company batch No. 11197753

FXA starting Material FXA purified in example 1 was diluted to a predetermined concentration with physiological saline

Coatron M4 semiautomatic blood coagulation Analyzer, TECO, Germany

3. Experimental methods

3.1 Effect of FXA raw Material on Normal coagulation quality control plasma aPPT

Reference to the aPTT test kit method, with minor modifications. And (3) placing the normal blood coagulation quality control blood plasma to room temperature, adding 1.0ml of deionized water into each blood plasma for dissolving, and placing the blood plasma at room temperature for 15min with shaking occasionally for standby. 0.01ml of FXA solution with different concentrations is taken, 0.09ml of standby normal blood coagulation quality control plasma is added, the mixture is uniformly mixed, preheated for 2min at 37 ℃, 0.1ml of aPTT reagent preheated at 37 ℃ is added, the heat preservation is continued for 3min, 0.1ml of calcium chloride preheated at 37 ℃ is added, and the aPTT time of the plasma is recorded. Deionized water was used as a blank instead of FXA solution.

3.2 Effect of FXA raw Material on plasma aPTT of poor factor

Reference to the aPTT test kit method, with minor modifications. And (3) placing the factor-poor plasma at room temperature, adding 1.0ml of deionized water into each factor-poor plasma for dissolving, and placing the factor-poor plasma at room temperature for 15min, wherein the factor-poor plasma is shaken occasionally and gently for standby. 0.01ml of FXA solution with different concentrations is taken, 0.09ml of spare factor-lacking plasma is added, the mixture is uniformly mixed, the temperature is kept for 2min at 37 ℃, 0.1ml of aPTT reagent preheated at 37 ℃ is added, the temperature is kept for 3min continuously, 0.1ml of calcium chloride preheated at 37 ℃ is added, and the aPTT time of the plasma is recorded. An equal volume of deionized water was added to the blank.

4. Results of the experiment

4.1 Effect of FXA raw Material on human Normal plasma aPTT

The effect of varying concentrations of FXA starting material on human normal plasma APTT time is shown in table 1.

FXA at 0.1,0.075,0.05,0.025,0.01U/ml significantly reduced aPTT in normal human plasma compared to the blank control and showed dose dependence (p < 0.05).

TABLE 1 Effect of FXA starting Material on human Normal plasma aPTT

Normal plasma was used as a blank control (con), FXA was administered at different concentrations, and the aPTT time was compared. P <0.001, compared to the blank control group; p <0.05, compared to the blank control group (n-4).

4.2 Effect of FXA raw Material on human factor VIII poor plasma aPTT

The effect of FXA at different concentrations on human factor VIII-poor plasma aPTT is shown in table 2. The aPTT time of the factor VIII-poor plasma group was significantly prolonged compared to human normal plasma (p < 0.001). The aPTT of factor VIII-poor plasma was significantly shortened and showed dose-dependence (p <0.001) after administration of different concentrations of FXA. Among them, aPTT of FXA group at 0.1U/ml was similar to the clotting time of normal plasma.

TABLE 2 Effect of FXA starting Material on human factor VIII poor plasma aPTT

The aPTT time was compared between normal plasma as a blank control (con), VIII-deficient plasma (VIII Free), and FXA at different concentrations. P <0.001, compared to the blank control group; # p <0.001, compared to factor VIII-poor plasma group (n-4).

4.3 Effect of FXA raw Material on human factor IX poor plasma aPTT

The effect of FXA at different concentrations on human factor IX-poor plasma aPTT is shown in table 3. The aPTT time was significantly prolonged in the factor IX-poor plasma group compared to human normal plasma (p < 0.001). Factor IX-poor plasma aPTT was significantly shortened and showed dose-dependence (p <0.01) after administration of different concentrations of FXA. Among them, aPTT of FXA group at 0.1U/ml was similar to the clotting time of normal plasma.

TABLE 3 Effect of FXA starting Material on human factor IX poor plasma aPTT

The aPTT time was compared between normal plasma as a control (con), IX-deficient plasma (IX Free), and FXA at different concentrations. P <0.001, compared to the blank control group; # p <0.001, # p <0.01, compared to factor IX-poor plasma group (n ═ 4).

5. Conclusion

In vitro experiments, the factor X activator can obviously shorten the partial thromboplastin time (aPTT) of normal plasma and factor VIII and factor IX-poor plasma, and shows dose dependence.

Comparative example 5 extraction of blood coagulation factor X activator from Viperi acutus venom

1. Dissolving snake venom:

dissolving 10g Vipera viper venom (purchased from Liaoning Tankoukang biopharmaceutical Co., Ltd.) in 100ml Tris-HCl buffer solution with pH of 8.0 and 0.05mol/L at 4-8 deg.C, centrifuging for 15min at 3000g, and collecting supernatant.

2. DEAE-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

2-2) loading and eluting: pumping the venom supernatant centrifuged in the step 2-1) into a balanced chromatographic column at a linear flow rate of 60cm/h, after the sample loading is finished, carrying out balanced elution by using Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L, a gradient eluent B is 300ml of Tris-HCl buffer solution with the pH value of 8.0 and 0.05mol/L and containing 0.5mol/L sodium chloride, the linear flow rate is 60cm/h, collecting each peak according to a 280nm wavelength ultraviolet detection map, and collecting 6 chromatographic peaks in total. The activity determination and gel electrophoresis tracing confirm that the 3 rd peak is the main peak of the activity of the factor X activator.

2-3) dialysis of the sample: and (3) dialyzing the phosphate buffer solution with pH6.0 and 0.05mol/L by using a membrane package with cut-off molecular weight of 10000Da for the active peak collected in the step 2-2), and fixing the volume of the sample to about 100 ml. Placing in a refrigerator at 4 ℃ for standby.

3. SP-Sepharose Fast Flow column chromatography: 2.6X 20cm (fillers from GE)

3-2) loading and eluting: pumping the snake venom solution dialyzed in the step 2 into a well balanced chromatographic column, carrying out linear flow velocity of 60cm/h, carrying out balanced elution by using phosphate buffer solution with pH of 6.0 and 0.05mol/L after the sample loading is finished, carrying out linear gradient elution after the ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is 300ml of phosphate buffer solution with pH of 6.0 and 0.05mol/L, a gradient eluent B is 300ml of phosphate buffer solution with pH of 6.0 and 0.05mol/L and containing 0.5mol/L sodium chloride, the linear flow velocity is 60cm/h, collecting each peak according to an ultraviolet detection map with the wavelength of 280nm, and collecting 5 chromatographic peaks in total. And determining the 3 rd peak as the main active peak of the X factor activator by activity measurement and gel electrophoresis tracking.

4. Lentin Lectin Sepharose 4B column chromatography 1.6X 20cm (Filler from GE)

4-2) loading and eluting: pumping the dialyzed snake venom solution obtained in the step 3 into a balanced chromatographic column, carrying out linear flow rate of 40cm/h, carrying out balanced elution by using Tris-HCl buffer solution with the pH value of 8.0 and 0.03mol/L, wherein the Tris-HCl buffer solution contains 0.8mol/L sodium chloride, 0.002mol/L calcium chloride and 0.002mol/L manganese chloride, carrying out linear gradient elution after ultraviolet absorption is reduced to a baseline, wherein a gradient eluent A is Tris-HCl buffer solution with the pH value of 8.0 and 0.03mol/L, the gradient eluent B is methyl-alpha-D-glucopyranoside with the pH value of 8.0 and the Tris-HCl buffer solution with the pH value of 0.03mol/L, carrying out linear flow rate of 40cm/h, collecting peaks according to a 280nm wavelength ultraviolet detection map, and collecting 3 chromatographic peaks in total. And determining the penetration peak as the main activity peak of the factor X activator by activity measurement and gel electrophoresis tracking.

5. DEAE-Sepharose Fast Flow column chromatography 1.6X 20cm (Filler from GE)

6. Superdex G75 column chromatography 2.6X 70cm (Filler from GE)

7. And (4) analyzing results:

7-1) purity detection

High performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, SEC-HPLC: G2000 WXL; RP-HPLC: C4 chromatographic column) and an electrophoresis system are adopted for detection, the SEC-HPLC detection shows 2 peaks, the RP-HPLC detection shows 2 peaks, and the contents of the blood coagulation factor X activator are 82% and 57% respectively according to the area normalization method. SDS-PAGE denaturing non-reducing electrophoresis detection of the target protein component shows multiple bands.

7-2) yield and specific activity

The activity detection of the target protein is carried out by an S2765 substrate method, the total activity of the coagulation factor X activator in 10g of dissolved solution is 70 ten thousand units, the activity collected after chromatographic purification is 8 ten thousand units, and the yield is about 11.4%. The specific activity is 987U/mg.

8. Conclusion

The method for extracting the blood coagulation factor X activator from Viper carinatus venom has the advantages that the yield is only 11.4 percent, the purity and the specific activity are low, the method is particularly suitable for extracting the venom blood coagulation factor X activator from the Viper carinatus venom, and meanwhile, the content of the venom blood coagulation factor X activator extracted by the method is high, and the method is more suitable for large-scale production.

Comparative example 6 extraction of factor X activator from Bothrops atrox venom

The extraction step comprises: (see Hofmann, H., Dumarey, C., Bon, C. (1983) Blood aggregation induced by Bothrops atrox vehicle identification and properties of a factor X activator. Biochimie 65(3): 201-210). )

1. Preparation of snake venom solution:

1.5g Bothrops atrox venom is dissolved in 30ml of 0.05mol/L phosphate buffer solution with pH7.5 in a chromatography cabinet at 4 deg.C, centrifuged at 8000rpm for 30min, and the supernatant is dialyzed with the same buffer solution to a volume of 40ml for use.

2. DEAE-cellulose column chromatography: 2.5X 60cm (DE52 Filler from GE)

2-1) DEAE-cellulose pre-column equilibration: 220ml of the packing was packed in a column, and the column volume was equilibrated by 10 or more volumes with 0.05mol/L phosphate buffer solution at pH 7.5.

2-2) loading, eluting and collecting: about 40ml of snake venom supernatant is pumped into a well balanced chromatographic column, 0.5ml/min, and the elution process is divided into three steps, 500ml of equilibrium solution is eluted → 1000ml of phosphate buffer solution containing 0-0.3mol/L sodium chloride and having pH7.5 and 0.05mol/L gradient elution → phosphate buffer solution containing 1mol/L sodium chloride and having pH7.5 and 0.05mol/L linear elution. Collecting each peak according to a 280nm wavelength ultraviolet detection spectrum, and collecting 7 chromatographic peaks in total. And (3) determining that the 4 th peak is the main peak of the activity of the factor X activator by the activity measurement of an S2765 substrate method and gel electrophoresis tracking.

2-3) sample freeze-drying dialysis: freeze-drying the active peak collected in the step 2-2), re-dissolving with 10ml of Tris-HCl buffer solution with pH7.5 and 5mmol/L, centrifuging 8000g for 15min to remove precipitate, dialyzing the re-dissolved solution, performing membrane package to cut off molecular weight of 10000Da, and freeze-drying after constant volume of 30ml for later use.

3. Sephadex g100(superfine) column chromatography: 2X 100cm (fillers from GE)

3-1) SephadexG100 column equilibration:

300ml of the packing is filled into a column, and the balance of Tris-HCL buffer solution with pH of 7.5 and 5mmol/L is more than 10 CV. The flow rate was 0.5 ml/min.

3-2) loading and eluting: and (3) re-dissolving the freeze-dried product in the step 2-3) by using 5ml of Tris-HCL buffer solution with the pH value of 7.5 and 5mmol/L, sucking 5ml of sample liquid by using an injector, loading the sample liquid, slowly adding the sample liquid along the side wall of the column in a rotating way, after the sample liquid completely enters the filler, rinsing the side wall and the gel surface by using less than 0.2ml of buffer solution, eluting at the flow rate of 0.5ml/min, collecting each peak according to an ultraviolet spectral width detection map under 280nm, and collecting 3 chromatographic peaks in total. And (3) determining that the 2 nd peak is the main peak of the activity of the factor X activator by using an S2765 substrate method activity measurement and gel electrophoresis tracking.

7. And (4) analyzing results:

7-1) purity detection

High performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, SEC-HPLC: G2000 WXL; RP-HPLC: C4 chromatographic column) and an electrophoresis system are adopted for detection, the SEC-HPLC detection shows a single peak, the RP-HPLC detection shows a single peak, and the contents of the blood coagulation factor X activator are 76% and 53% respectively according to the area normalization method. SDS-PAGE denaturing non-reducing electrophoresis detection of the target protein component shows 4 bands.

7-2) yield and specific activity

The activity detection of the target protein is detected by an S2765 substrate method, the total activity of the blood coagulation factor X activator in 1.5g of dissolved solution is 9 ten thousand units, the activity collected after chromatographic purification is 2.25 ten thousand units, and the yield is 25%. The specific activity is 876U/mg.

7. And (4) conclusion:

the reports of separating the blood coagulation factor X activator from the agkistrodon spearhead venom are few, domestic documents and patent reports mostly extract the blood coagulation factor X activator from the agkistrodon acutus venom, and experimental literature methods extract the blood coagulation factor X activator from the agkistrodon spearhead venom, so that the yield is higher by 25%, but the purity and the specific activity are low, and the method in the literature is not suitable for preparing the high-purity blood coagulation factor X activator in a large scale.

Sequence listing

<110> Liaoning Yuandaokang biopharmaceutical Co., Ltd

<120> a blood coagulation factor X activator and a preparation method thereof

<130> DIC16110057

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> H chain N-terminal sequence of factor X activator

<400> 1

Leu Thr Pro Glu Gln Gln Ala Tyr Leu Asp Ala Lys Lys Tyr Val

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> N-terminus of L1 chain of factor X activator

<400> 2

Asp Cys Pro Ser Asp Trp Ser Ser Tyr Glu Gly His Cys Tyr Arg

1 5 10 15

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> N-terminus of L2 chain of factor X activator

<400> 3

Asp Cys Asn Ser Tyr Trp Ser Thr Tyr Glu Gly Arg Ser Tyr Arg

1 5 10 15

Claims

1. A method of preparing a factor x activator, the method comprising the steps of:

1) dissolving Agkistrodon halys venom in 0.01-0.1mol/L alkaline buffer solution with pH of 7.4-8.5 to obtain solution with concentration of 20-150mg/ml, centrifuging, and collecting supernatant;

2) carrying out anion exchange chromatography and linear gradient elution on the supernatant obtained in the step 1), collecting eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 10000Da and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an alkaline buffer solution with pH of 7.4-8.5 and concentration of 0.04-0.06 mol/L; the gradient eluent B is an alkaline buffer solution which contains 0.4 to 0.6mol/L NaCl, has the pH value of 7.4 to 8.5 and has the concentration of 0.04 to 0.06 mol/L;

wherein, the anion exchange chromatography filler is DEAE-Sepharose Fast Flow;

3) performing cation exchange chromatography and linear gradient elution on the eluent dialyzed in the step 2), collecting the eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 10000Da and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an acidic buffer solution with pH of 5.5-6.5 and concentration of 0.04-0.08 mol/L; the gradient eluent B is an acidic buffer solution which contains 0.4-0.6mol/L NaCl, has pH of 5.5-6.5 and has concentration of 0.04-0.08 mol/L;

wherein, corresponding to the next step of affinity chromatography or composite filler chromatography, the dialysate is selected from affinity chromatography dialysate or composite filler chromatography dialysate, and the affinity chromatography dialysate contains 0.3-0.8mol/LNaCl and 0.002-0.005mol/LCaCl₂And MnCl₂An alkaline buffer solution with pH of 7.5-8.0 and concentration of 0.03-0.05 mol/L; the chromatography dialysate of the composite filler is a solution containing 0.3mol/L sodium chloride,An alkaline buffer solution with pH of 8.0 and concentration of 0.03 mol/L;

wherein the cationic filler is SP-Sepharose Fast Flow;

4) carrying out affinity chromatography or composite filler chromatography on the eluent dialyzed in the step 3), carrying out linear gradient elution, collecting the eluent containing the active elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 10000Da and dialysate;

wherein, the solid phase filler of the affinity chromatography is selected from an affinity filler Lentin Lectin Sepharose 4B and ConA Sepharose 4B containing two types of ligands; the composite filler for chromatography of the composite filler is CHT ceramic hydroxyapatite I;

wherein, in the linear gradient elution of the affinity chromatography, a gradient eluent A is an alkaline buffer solution containing 0.2-1mol/L NaCl, the pH value is 7.6-8.4 and the concentration is 0.02-0.05 mol/L; the gradient eluent B is an alkaline buffer solution with pH of 7.6-8.4 and concentration of 0.02-0.05mol/L, which contains 0.1-1mol/L NaCl and 0.1-0.5mol/L saccharide competitive eluent;

in the linear gradient elution of the composite filler chromatography, a gradient eluent A is an alkaline buffer solution which contains 0.3mol/L NaCl and has the pH value of 8.0 and the concentration of 0.03mol/L, and a gradient eluent B is an alkaline buffer solution which contains 0.8mol/L NaCl and has the pH value of 8.0 and the concentration of 0.03 mol/L;

wherein the dialysate is an alkaline buffer solution which contains 0.05-0.1mol/L NaCl, has pH of 7.5-8.5 and concentration of 0.02-0.05 mol/L;

5) carrying out anion exchange chromatography and linear gradient elution on the eluent dialyzed in the step 4), collecting the eluent containing the activity elution peak of the blood coagulation factor X activator, and dialyzing the eluent by using a membrane package with the molecular weight cutoff of 10000Da and dialysate;

wherein, in the linear gradient elution, the gradient eluent A is an alkaline buffer solution which contains 0.05-0.1mol/L NaCl, has the pH value of 7.5-8.5 and has the concentration of 0.02-0.05 mol/L; the gradient eluent B is an alkaline buffer solution which contains 0.4-0.6mol/L NaCl, has the pH value of 7.5-8.5 and has the concentration of 0.02-0.05 mol/L;

wherein the dialysate is an alkaline buffer solution containing 0.05-0.1mol/L NaCl, with pH of 7.5-8.0 and concentration of 0.01-0.1 mol/L;

wherein the anion exchange chromatography packing is selected from DEAE-Sepharose Fast Flow;

6) performing gel Superdex G75 filtration chromatography on the dialyzed eluate, performing linear elution, and collecting the eluate containing the active elution peak of the blood coagulation factor X activator to obtain the blood coagulation factor X activator;

wherein the eluent is an alkaline buffer solution containing 0.05-0.1mol/L NaCl, with the pH value of 7.5-8.0 and the concentration of 0.01-0.1 mol/L.

2. The method according to claim 1, wherein in step 2), the dialyzed eluate is stored at 4 ℃.

3. The method according to claim 1, wherein in step 3), the dialyzed eluate is stored at 4 ℃.

4. The method according to claim 1, wherein in step 4), the dialyzed eluate is stored at 4 ℃.

5. The method of claim 1, wherein said collecting the eluate containing the elution peak of factor X activator activity is effected by a method comprising:

collecting eluate, performing ultraviolet spectrophotometer detection on the eluate at 280nm wavelength, collecting each elution peak according to ultraviolet absorption spectrum and determining activity, performing SDS-PAGE gel electrophoresis tracking detection, and collecting eluate containing active elution peak of blood coagulation factor X activator.

6. The method of claim 5, wherein an S-2765 substrate chromogenic assay is used in the activity assay.

7. The method according to claim 1, wherein in step 1), the pH of the alkaline buffer is 8.0 and the concentration is 0.05 mol/L.

8. The method according to claim 1, wherein in step 1), the centrifugation conditions are: at 4-8 deg.C, the relative centrifugal force is 1200-20000 g, and the centrifugal time is 5-20 min.

9. The method according to claim 8, wherein the relative centrifugal force is 3000g to 4000g and the centrifugation time is 15 min.

10. The method according to claim 1, wherein in step 2), the gradient eluent A is an alkaline buffer solution with a pH of 8.0 and a concentration of 0.05mol/L, and the gradient eluent B is an alkaline buffer solution with a pH of 8.0 and a concentration of 0.05mol/L and containing 0.5mol/L NaCl.

11. The method according to claim 1, wherein in step 2), 10 column volumes or more are equilibrated with an alkaline buffer solution having a pH of 7.4 to 8.5 and a concentration of 0.04 to 0.06mol/L as an equilibration solution before anion exchange chromatography is performed.

12. The method according to claim 11, wherein the equilibration solution is an alkaline buffer solution with a pH of 8.0 and a concentration of 0.05 mol/L.

13. The method according to claim 12, wherein in step 2), the linear flow rate at equilibrium is 60-200 cm/h.

14. The method according to claim 13, wherein in step 2), at equilibrium, the linear flow rate is 100 cm/h.

15. The method according to claim 1, wherein in step 2), the linear flow rate at elution is 20 to 100 cm/h.

16. The method according to claim 1, wherein in step 2), the linear flow rate is 60cm/h at the time of elution.

17. The method according to claim 1, wherein in step 2), the dialysate is an acidic buffer with a pH of 5.5-6.5 and a concentration of 0.04-0.08 mol/L.

18. The method according to claim 1, wherein in step 2), the dialysate is an acidic buffer solution with a pH of 6.0 and a concentration of 0.05 mol/L.

19. The method according to claim 1, wherein in step 3), the gradient eluent A is an acidic buffer solution with pH of 6.0 and a concentration of 0.05 mol/L; the gradient eluent B is an acidic buffer solution which contains 0.5mol/L NaCl, has the pH value of 6.0 and has the concentration of 0.05 mol/L.

20. The method according to claim 1, wherein in step 3), 10 column volumes or more are equilibrated with an acidic buffer solution having a pH of 5.5 to 6.5 and a concentration of 0.04 to 0.08mol/L as an equilibration solution before the cation exchange chromatography is performed.

21. The method according to claim 20, wherein the equilibration solution is an acidic buffer solution with a pH of 6.0 and a concentration of 0.05 mol/L.

22. The method as claimed in claim 21, wherein in step 3), the linear flow rate at equilibrium is 60-200 cm/h.

23. The method according to claim 22, wherein in step 3), the linear flow rate at equilibrium is 100 cm/h.

24. The method according to claim 1, wherein in step 3), the linear flow rate at elution is 20 to 100 cm/h.

25. The method according to claim 1, wherein in step 3), the linear flow rate is 60cm/h at the time of elution.

26. The method of claim 1, wherein in step 3), the affinity chromatography dialysate is 0.8mol NaCl/L, 0.002mol/LCaCl₂And MnCl₂An alkaline buffer solution with pH of 8.0 and concentration of 0.03 mol/L; the chromatography dialysate of the composite filler is an alkaline buffer solution containing 0.3mol/L sodium chloride, with the pH value of 8.0 and the concentration of 0.03 mol/L.

27. The method of claim 1, wherein in step 4), the gradient eluent A is an alkaline buffer solution containing 0.8mol NaCl/L, pH at 8.0 and having a concentration of 0.03mol/L in the affinity chromatography; the gradient eluent B is an alkaline buffer solution with the pH value of 8.0 and the concentration of 0.03mol/L, and contains 0.8mol/L NaCl and 0.2-0.4mol/L carbohydrate competitive eluent.

28. The method according to claim 1, wherein in step 4) the saccharide-type competitive eluent is selected from the group consisting of methylglucose-type competitive eluents and methylglucane-type competitive eluents.

29. The method of claim 1, wherein in step 4), 0.2-1mol/L NaCl, 0.001-0.003mol/L CaCl is used before the affinity chromatography₂And MnCl₂And an alkaline buffer solution having a pH of 7.6 to 8.4 and a concentration of 0.02 to 0.05mol/L as an equilibrium solution to equilibrate for 10 column volumes or more.

30. The method of claim 29, wherein the equilibration solution is 0.8mol/L NaCl, 0.002mol/LCaCl₂And MnCl₂An alkaline buffer solution having a pH of 8.0 and a concentration of 0.03 mol/L.

31. The method according to claim 1, wherein in step 4), 10 column volumes or more are equilibrated with an alkaline buffer solution containing 0.2 to 0.4mol/L NaCl, having a pH of 7.6 to 8.4 and a concentration of 0.02 to 0.05mol/L, as an equilibration solution before the composite filler chromatography is performed.

32. The method of claim 31, wherein the equilibration solution is 0.03mol/L alkaline buffer solution containing 0.3mol/L NaCl and having a pH of 8.0.

33. The method of claim 30, wherein in step 4), the linear flow rate at the time of the affinity chromatography equilibrium is 30-70 cm/h.

34. The method of claim 33, wherein in step 4), the linear flow rate at the time of the affinity chromatography equilibrium is 60 cm/h.

35. The method according to claim 1, wherein in step 4), the linear flow rate at elution of affinity chromatography is 30-70 cm/h.

36. The method of claim 1, wherein in step 4), the linear flow rate for elution by affinity chromatography is 40 cm/h.

37. The method as claimed in claim 32, wherein in step 4), the linear flow rate of the composite packing at chromatographic equilibrium is 50-200 cm/h.

38. The method as claimed in claim 37, wherein in step 4), the linear flow rate of the composite packing at chromatographic equilibrium is 100 cm/h.

39. The method according to claim 1, wherein in step 4), the linear flow rate of the composite chromatography elution is 50-100 cm/h.

40. The method of claim 1, wherein in step 4), the linear flow rate of the composite chromatography elution is 80 cm/h.

41. The method according to claim 1, wherein in step 4), the dialysate is an alkaline buffer solution containing 0.1mol/L NaCl at pH8.0 and a concentration of 0.03 mol/L.

42. The method according to claim 1, wherein in step 5) the gradient eluent a is an alkaline buffer containing 0.1mol of NaCl at a pH of 8.0 and a concentration of 0.03 mol/L; the gradient eluent B is an alkaline buffer solution with 0.03mol/L concentration and pH of 8.0 and containing 0.5mol/L NaCl.

43. The method according to claim 1, wherein in step 5), 10 column volumes or more are equilibrated with an alkaline buffer solution containing 0.05 to 0.1mol/L of NaCl at a pH of 7.5 to 8.5 and having a concentration of 0.02 to 0.05mol/L as an equilibration solution before the anion exchange chromatography is performed.

44. The method according to claim 43, wherein the equilibration solution is an alkaline buffer solution with 0.03mol/L and pH8.0, containing 0.1mol/L NaCl.

45. The method as claimed in claim 44, wherein in step 5), the linear flow rate at equilibrium is 60-200 cm/h.

46. The method of claim 45, wherein in step 5), the linear flow rate at equilibrium is 100 cm/h.

47. The method according to claim 1, wherein in step 5), the linear flow rate at elution is 20 to 100 cm/h.

48. The method according to claim 1, wherein in step 5), the linear flow rate at elution is 60 cm/h.

49. The method according to claim 1, wherein in step 5), the dialysate is an alkaline buffer solution containing 0.05mol/L NaCl at pH8.0 and a concentration of 0.02 mol/L.

50. The method according to claim 1, wherein in step 6), the eluent is an alkaline buffer solution containing 0.05mol/L NaCl and having a pH of 8.0 and a concentration of 0.02 mol/L.

51. The method according to claim 1, wherein in step 6), 5 column volumes or more are equilibrated with an alkaline buffer solution containing 0.01 to 0.05mol/L of NaCl, having a pH of 7.5 to 8.5 and having a concentration of 0.01 to 0.05mol/L, as an equilibration solution, before performing the gel filtration chromatography.

52. The method according to claim 51, wherein the equilibration solution is an alkaline buffer solution with 0.02mol/L and pH8.0, containing 0.05mol/L NaCl.

53. The method as claimed in claim 52, wherein in step 6), the linear flow rate at equilibrium is 20-80 cm/h.

54. The method of claim 53, wherein in step 6), the linear flow rate at equilibrium is 60 cm/h.

55. The method according to claim 1, wherein in step 6), the linear flow rate at elution is 20 to 60 cm/h.

56. The method according to claim 1, wherein in step 6), the linear flow rate at elution is 40 cm/h.

57. The method as claimed in claim 1, wherein in step 6), the separation range of the gel packing is 3000-300000 Da.

58. The method of any one of claims 1-57, wherein the alkaline buffer is selected from the group consisting of Tris-HCl buffer, phosphate buffer, and phosphate-citrate buffer;

the acidic buffer is selected from phosphate buffer, phosphate-citric acid buffer and acetate buffer.