Method for purifying hemagglutinin of spearhead agkistrodon halys

A purification method and hemocoagulase technology, applied in the field of hemocoagulase purification, can solve the problems of low specific activity of pure hemocoagulase, deviation of hemocoagulase purity, inconvenient large-scale production, etc., and achieve consistent product quality Good sex, avoid immunogenic reaction, low protein effect

Pending Publication Date: 2022-07-01
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This shows that the purity of the hemocoagulase obtained in the patent application is biased
[0008] (2) The stability between batches is poor, and different batches of snake venom have different results
[0011] (3) The specific activity is low, the specific activity of the hemocoagulase pure product obtained using the method is low, only 1200-1600IU / mg
[0012] In summary, the methods for extracting snake venom hemagglutinin disclosed in literature or patent applications in the prior art are difficult to meet the ideal purification requirements and are not convenient for large-scale production

Method used

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  • Method for purifying hemagglutinin of spearhead agkistrodon halys
  • Method for purifying hemagglutinin of spearhead agkistrodon halys
  • Method for purifying hemagglutinin of spearhead agkistrodon halys

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Purification of hemagglutinin using the method of the present invention

[0071] 1. Dissolve 5g of the venom of the spearhead pit viper in 50mL of 20mM Tris-HCl, 0.2M NaCl, pH8.0 buffer, soak it at room temperature to fully dissolve it, and then centrifuge at 5000rpm for 5min to remove the precipitate and recover the supernatant .

[0072] 2. Benzamidine Sepharose 6B affinity chromatography column (2.6cm×50cm) was equilibrated for 3 column volumes with 20mM Tris-HCl, 0.2M NaCl, pH8.0 buffer, and then centrifuged The supernatant of the snake venom was loaded onto the chromatography column; after the loading, the buffer was continuously washed for 3 column volumes, and eluted with an eluent of 20 mM Tris-HCl, 0.2 M NaCl, 1 M arginine, pH 8.0 , collect the target components.

[0073] 3. The target component after affinity chromatography was ultrafiltered and exchanged into a buffer of 20 mM PB, pH 6.0, and the molecular weight cut-off of the ultrafiltration mem...

Embodiment 2

[0078] Example 2 Purification of hemagglutinin using the method of the present invention

[0079] 1. Dissolve 5 g of the venom from the venom of the spearhead pit viper in 30 mL of 50 mM Tris-HCl, 0.1 M NaCl, pH 8.0 buffer, soak it at room temperature to fully dissolve it, and then centrifuge at 5000 rpm for 5 min to remove the precipitate and recover the supernatant. .

[0080] 2. Benzamidine Sepharose 6B affinity chromatography column (2.6cm×50cm) was equilibrated for 3 column volumes with 50mM Tris-HCl, 0.1M NaCl, pH7.0 buffer, and then centrifuged The supernatant of the snake venom was loaded onto the chromatography column; after the loading, continued to wash with buffer for 3 column volumes, and eluted with the eluent of 50mM Tris-HCl, 0.1M NaCl, 0.2M arginine, pH 6.0 , collect the target components.

[0081] 3. The target component after affinity chromatography was ultrafiltered and exchanged into a buffer of 5mM PB, pH 6.2, and the molecular weight cut-off of the ult...

Embodiment 3

[0086] Example 3 Purification of hemagglutinin using the method of the present invention

[0087] 1. Dissolve 5 g of the venom of the spearhead pit viper in 60 mL of 100 mM Tris-HCl, 0.5 M NaCl, pH 9.0 buffer, soak it at room temperature to fully dissolve it, and then centrifuge at 5000 rpm for 5 min to remove the precipitate and recover the supernatant. .

[0088] 2. Benzamidine Sepharose 4B affinity chromatography column (2.6cm×50cm) was equilibrated for 3 column volumes with 100mM Tris-HCl, 0.5M NaCl, pH9.0 buffer, and then centrifuged The supernatant of snake venom was loaded onto the chromatography column; after the loading, continued to wash with buffer for 3 column volumes, and eluted with the eluent of 100mM Tris-HCl, 0.5M NaCl, 0.05M arginine, pH 9.0 , collect the target components.

[0089] 3. The target component after affinity chromatography was ultrafiltered and exchanged into a buffer of 50 mM PB, pH 6.1, and the molecular weight cut-off of the ultrafiltration ...

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Abstract

The invention provides a method for purifying agkistrodon spearhead hemocoagulase, which comprises the following steps: pretreating agkistrodon spearhead venom, and sequentially carrying out benzamidine agarose gel affinity chromatography, cation exchange chromatography and hydrophobic chromatography to obtain the agkistrodon spearhead hemocoagulase. According to the method provided by the invention, the process steps are further simplified, the purification period of the hemocoagulase is greatly shortened, the process environment exposure risk is reduced, the process stability is high, the product quality consistency is good, and the clinical medication safety is facilitated.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for purifying hemagglutinin extracted from the venom of the spearhead pit viper. Background technique [0002] Coagulation is a complex physiological process, which is roughly divided into three stages: the first stage, the activation of coagulation factor X (FX); the second stage, the activation of prothrombin; the third stage, the conversion of fibrinogen to fibrin . [0003] The extraction of hemostatic agents from snake venom has a long history. At present, the most successful clinical application is Reptilase, a hemostatic drug produced in Switzerland, which immediately stops bleeding. Its main component is Snake Venom Thrombin-like Enzyme (SVTLE) extracted from the venom of the Brazilian snake (Bothrops Jrarace, Lachesisatrox). The enzyme belongs to the trypsin family of serine proteases with arginine esterase and amidase activities. The enzyme can hydrol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/04
CPCC12N9/6418A61P7/04C12Y304/21005A61K38/00
Inventor 李秀琳陈颖李怀超李雪龙李佳杨薛百忠
Owner GRAND LIFE SCI (LIAONING) CO LTD
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