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A method for extracting blood coagulation factor X activator from snake venom

A blood coagulation factor and activator technology, applied in the field of biological extracts, can solve the problems of high economic cost, few steps, cumbersome operation, etc.

Active Publication Date: 2021-04-16
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are few reports on the direct isolation and purification of specific coagulation factor X activators from snake venom
Chinese patent application CN1249338A records the use of Molo Q column for rapid protein liquid chromatography to extract blood coagulation factor X activating enzyme from Russell's vipervenom toxin. This method has few steps and can meet the needs of research and development, but it is too simple Purification step does not meet the requirements of the pharmaceutical industry
[0006] Chinese patent application CN101104847A records the activation of blood coagulation factor X from Viper venom through DEAE-SehadexA-50 ion exchange chromatography, dialysis, cellulose DEAE-32 ion exchange chromatography and Superose-12 gel filtration chromatography Enzyme method; the dialysis step is used in this process, the dialysis treatment time is long, the operation is cumbersome, and it is not suitable for large-scale production
[0007] Chinese patent application CN102242102A records that the coagulation factor X activator active protein is obtained from Agkistrodon lanceolata venom through DEAE-Sepharose FastFlow ion exchange chromatography, dialysis, cellulose DEAE-52 ion exchange chromatography and Sephadex-G75 gel filtration chromatography, But in this method, 10g snake venom dry powder is 34mg after purification harvest protein, and yield is only 0.34%, and this method yield is lower, and economic cost is higher

Method used

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  • A method for extracting blood coagulation factor X activator from snake venom
  • A method for extracting blood coagulation factor X activator from snake venom
  • A method for extracting blood coagulation factor X activator from snake venom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: Preparation of snake venom raw material liquid

[0093] Take about 10ml of viper venom (Guangxi) (protein concentration is about 260mg / ml), dilute it with 20mM Tris-HCl solution (pH8.5) to a concentration of 26mg / ml, centrifuge at 12000×g for 20 minutes at 4°C, collect supernatant.

Embodiment 2

[0094] Example 2: Virus inactivation

[0095] Adopt S / D method to carry out virus inactivation to the snake venom raw material liquid that embodiment 1 obtains, concrete steps are as follows:

[0096] After the supernatant collected in Example 1 was filtered with a 0.45 μm filter membrane, Tween-80 and tributyl phosphate were added successively so that the final volume concentration of Tween-80 was 1%, and the final volume concentration of tributyl phosphate was 0.3 %. After thorough mixing, the mixture was placed in a water bath at 25°C±2°C for 2 hours to inactivate the virus.

Embodiment 3

[0097] Embodiment 3: anion exchange chromatography

[0098] Q Sepharose HP chromatography column was equilibrated with 20mM Tris-HCl (pH8.5) buffer for 3 column volumes before use.

[0099] The snake venom raw material solution obtained through virus inactivation obtained in Example 2 is loaded with a flow rate of 90 cm / hour. After sample loading, wash with 20mM Tris-HCl (pH8.5) solution to the baseline at a flow rate of 90cm / hour.

[0100] Wash with 20mM Tris-HCl-0.2M NaCl (pH8.5) solution, flow rate 90cm / hour.

[0101] After the impurity washing is completed, 20%-100% 20mM Tris-HCl-0.2M NaCl (pH8.5) solution is used to elute for 10 column volumes at a flow rate of 90cm / hour. When the conductance increased to 10ms / cm, the target peak began to appear, and the components corresponding to the target peak were collected. Stop collecting the components corresponding to the target peak until the target peak drops to the baseline.

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Abstract

The invention provides a method for extracting coagulation factor X activator from snake venom, and the use of the coagulation factor X activator extracted by the method in preparing hemostatic medicine and medicine for treating bleeding diseases. The method of the present invention comprises a hydroxyapatite column chromatography step, wherein the chromatographic conditions are as follows: the filler is a type I or type II hydroxyapatite filler, and the eluent is a phosphate buffer containing NaCl with a pH value of 6.5-7.5 , wherein the concentration of phosphate buffer is 10mM-30mM; the concentration of NaCl is 0.1M-0.8M. The method of the present invention uses snake venom as a raw material, and for the first time applies the hydroxyl phosphorus chromatography method to the method for large-scale extraction of blood coagulation factor X activator. The final product obtained by the method of the invention has high specific activity, high purity and high yield; no dialysis operation, stable process and suitable for large-scale production; the chromatographic column can be used repeatedly, saving cost.

Description

technical field [0001] The invention relates to the technical field of biological extracts, in particular to a method for extracting coagulation factor X activator from snake venom. Background technique [0002] The human coagulation mechanism is a very complex and delicate process. So far, 12 factors have been found to be involved in coagulation. Among them, coagulation factor X is one of the very key components in the coagulation system. Coagulation factor X activator activates coagulation factor X at the site of blood vessel damage to promote thrombin generation. Under the action of thrombin, fibrinogen soluble in plasma is converted into fibrin monomer; at the same time, thrombin activates XIII to XIIIa, making fibrin monomers interconnected to form water-insoluble fibrin polymers, And intertwine with each other to form a net to enclose blood cells and form blood clots to achieve the purpose of rapid hemostasis. [0003] There are many enzymes in snake venom that can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/04
Inventor 张世贤李贺平
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD