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Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method

A technology of hemocoagulase and Agkistrodon, which is applied in the field of separation and purification to obtain hemocoagulase and its preparation, to achieve the effects of stable preparation process, shortened coagulation time and low toxicity

Inactive Publication Date: 2009-06-03
沈居仁 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In 1936, Klobusizky and konig isolated an enzyme protein that can shorten the coagulation time from the venom of the willow-leaf viper, named Coagulase, and many scholars successively confirmed the existence of this enzyme in different Agkistrodon halys. In the 1960s Since then, with the improvement of protein purification and separation technology and the in-depth research of biochemistry and pharmacology, the great value of hemocoagulase in clinical hemostasis application has been determined. Among them, Reptilase is a product of Swiss Solco Basle Ltd. Bothrope jararaca) snake venom is extracted from the hemostatic agent with a molecular weight of about 31KD single-chain hemocoagulase. In addition to hemocoagulase, its components also contain hemocoagulase-like kinase, which activates pro-hemocoagulase into hemocoagulase , to promote the conversion of fibrinogen into fibrin, and form the same coagulation effect as hemocoagulase. ("Enzymatic hemostatic injection to stop bleeding" Solco Basle Ltd properties) The hemostatic agent has a definite curative effect, but in our country, there is no Brasilia lancehead Distribution. In order to obtain hemocoagulase from domestic snake species of the same genus, domestic scholars have made unremitting efforts. For example, using the snake venom thrombin of Agkistrodon acutus distributed in various provinces in South China to develop, application number 02138630.7 invention patent publication "Akistrodon akistrodon venom thrombin with hemostatic activity" and application number 03140154.6 invention patent publication "Akikikikiki venom thrombin used as a drug for treating hemorrhagic diseases" successively separated thrombin components, and they N - There are differences in the terminal amino acid sequence, and both are composed of two subunits, A and B, with a specific activity of about 120U / mg

Method used

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  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method
  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method
  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, 20060418 batch of Agkistrodon acutus venom 3.6KD hemagglutinin production example

[0032] Weigh 10g of the lyophilized product of Agkistrodon acutus venom with 50ml of sterile water for injection, dialyzed overnight in deionized water at 4℃, centrifuge at 8000rpm, 15min, 4℃, and adjust the supernatant to 3.5 with 1M Tris-HCl (PH8.0). After ms / cm, load the affinity chromatography column Metal Sepharose FF, 1M NH, which is pre-equilibrated with 0.05M Tris-HCl (PH8.0) 4 Cl carries out phase elution and collects the breakthrough peak; the balanced DEAE-Sepharose column on the breakthrough peak, 0.05M Tris-HCl (PH8.0) zero wash 1 times the column bed volume, use 0.05M Tris-HCl+1.0 M NaCl (PH8.0) was used for gradient elution. Five peaks (ie crude hemagglutinin fraction) were collected under UV detection at 280nm, and the active peak was desalted and lyophilized. The lyophilized product was dissolved in 10-20ml 0.05M Tris- HCl(PH7.5)+0.1M KCl, pre-equilibrated with 0....

Embodiment 2

[0033] Example Two, 20060428 batch of Agkistrodon acutus venom 3.6KD hemagglutinin production example

[0034] Weigh 10g of the lyophilized product of Agkistrodon acutus venom with 50ml of sterile water for injection, dialyzed overnight in deionized water at 4℃, centrifuge at 8000rpm, 15min, 4℃, and adjust the supernatant to 3.5 with 1M Tris-HCl (PH8.0). After ms / cm, load the affinity chromatography column Metal Sepharose FF column, 1M NH, which is pre-equilibrated with 0.05M Tris-HCl (PH8.0) 4 Cl carries out phase elution, and collects the breakthrough peak; the DEAE-Sepharose column is well balanced on the breakthrough peak, and after 0.05M Tris-HCl (PH8.0) zero wash 1 column bed volume, use 0.05M Tris-HCl+1.0 M NaCl (PH8.0) was used for gradient elution, 5 peaks (ie crude hemagglutinin fraction) were collected under UV 280nm detection, and the active peak was desalted and lyophilized. The lyophilized product is dissolved in 10-20ml 0.05M Tris-HCl(PH7.5)+0.1M KCl, and the Superd...

Embodiment 3

[0035] Example three, 20060523 batch of Agkistrodon acutus venom 3.6KD hemagglutinin production example

[0036]Weigh 10g head of the lyophilized Agkistrodon acutus venom with 50ml sterile water for injection, dialyze overnight in deionized water at 4°C, centrifuge at 8000rpm, 15min, 4°C, and adjust the supernatant with 1M Tris-HCl (PH8.0) to adjust the conductivity to After 3.3ms / cm, load the Metal Sepharose F.FXK50 / 30 (product of GE Healthcare), 1M NH, which has been pre-balanced with 0.05MTris-HCl (PH8.0) 4 Cl carries out phase elution, and collects the breakthrough peak; the DEAE-Sepharose column is well-balanced on the breakthrough peak, and after 0.05M Tris-HCl (Ph8.0) zero wash 1 column bed volume, use 0.05M Tris-HCl+1.0 M NaCl (PH8.0) is used for gradient elution. 5 elution peaks (ie crude hemagglutinin fraction) are collected under UV 280nm detection, and the activity peak is desalted and lyophilized. The lyophilized product is dissolved in 10-20ml 0.05M Tris-HCl (PH7.5)+...

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Abstract

The present invention is Agkistrodon acutus venon 36KD single-stranded hemocoagulase and its preparation process, and aims at providing single-stranded Agkistrodon acutus venon hemocoagulase with high potency and low toxicity. SDS-PAGE electrophoresis shows that the hemocoagulase has molecular weight of 36KD+ / -2KD, single color zone, and at least 90 % homogeneity with the N end sequence of the single-stranded amino acid shown in SEQ ID No. 1. The preparation process includes dissolving Agkistrodon acutus venon in Tris-HCl buffering solution (pH8.0) and dialysis; adding the supernatant to Metal Sepharose F.F affinity chromatographic column and collecting the penetrating peak component; adding the penetrating peak component to DEAE-Sepharose F.F ion exchange column and collecting active peak component; separating in Superdex 75 column and Sephacryl S-100 column; desalting in Sephadex G-25 column; sterilizing and freeze drying.

Description

Technical field [0001] The present invention relates to an enzyme and a method for separating and purifying the enzyme, particularly a method for separating and purifying hemagglutinin from Agkistrodon acutus and its preparation. Background technique [0002] In 1936, Klobusizky and konig isolated an enzyme protein that can shorten the clotting time from the venom of the willow viper snake and named it Coagulase. Many scholars have successively confirmed the existence of this enzyme in different vipers in the subfamily. 1960s Since then, with the improvement of protein purification and separation technology and the in-depth research of biochemistry and pharmacology, the great value of hemocoagulase in the clinical application of hemostasis has been determined. Among them, Reptilase is the Swiss Solco Basle Ltd. Bothrope jararaca) is a hemostatic agent prepared from snake venom with a molecular weight of about 31KD and single-chain hemagglutinin. In addition to hemagglutinin, its ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74
Inventor 沈居仁郑颖范泉水
Owner 沈居仁
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