Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method

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 ...

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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

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