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Method for extracting thrombin-like enzyme from Agkistrodon halys ussuriensis snake venom

A technology similar to thrombin and Agkistrodon halys, applied in the field of medicine, can solve problems such as low yield, poor purity, and long production cycle, and achieve the effects of short production cycle, stable process, and simple operation

Inactive Publication Date: 2011-11-16
辽宁远大诺康医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this process method, the production cycle is long, the yield is low, and the purity is poor

Method used

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  • Method for extracting thrombin-like enzyme from Agkistrodon halys ussuriensis snake venom
  • Method for extracting thrombin-like enzyme from Agkistrodon halys ussuriensis snake venom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The steps for extracting thrombin-like enzyme from Agkistrodon halys venom are as follows:

[0022] (1) Take 30g of white-browed snake venom dry powder (batch number 070704), dissolve it in 200ml of PBS buffer solution I with pH 6.0, soak and dissolve in a refrigerator at 4°C, overnight, centrifuge at 4500 rpm for 15 minutes, remove the precipitate, and collect snake venom supernatant;

[0023] (2), SP Sepharose Fast Flow cation exchange chromatography column (2.6cm×30cm) equilibrate at least 5 column volumes with PBS buffer solution of pH 6.0, put the above supernatant on the column, control the flow rate at 3.0ml / min, After the sample is finished, the column is equilibrated with 200ml buffer;

[0024] (3) Elute linearly with PBS buffer solution II containing 0.2M NaCl at pH 6.0, detect with an ultraviolet detector at 280 nm, collect activity peaks, and obtain a crude thrombin-like extract;

[0025] (4) The thrombin-like crude extract needs to be dialyzed at least th...

Embodiment 2

[0028] The steps for extracting thrombin-like enzyme from Agkistrodon halys venom are as follows:

[0029] (1) Take 30g of white-browed snake venom dry powder (batch number 090907), dissolve it in 150ml of PBS buffer solution I with pH 6.0, soak and dissolve in a refrigerator at 4°C, overnight, centrifuge at 4500 rpm for 15 minutes, remove the precipitate, and collect snake venom supernatant;

[0030] (2), SP Sepharose Fast Flow cation exchange chromatography column (2.6cm×30cm) equilibrate at least 5 column volumes with PBS buffer solution of pH 6.0, put the above supernatant on the column, control the flow rate at 3.0ml / min, After the sample is finished, the column is equilibrated with 200ml buffer;

[0031] (3) Elute linearly with PBS buffer solution II containing 0.2M NaCl at pH 6.0, detect with an ultraviolet detector at 280 nm, collect activity peaks, and obtain a crude thrombin-like extract;

[0032] (4) The thrombin-like crude extract needs to be dialyzed at least th...

Embodiment 3

[0035] The steps for extracting thrombin-like enzyme from Agkistrodon halys venom are as follows:

[0036] (1) Take 30g of white-browed snake venom dry powder (batch number 100331), dissolve it in 200ml of PBS buffer solution I with pH 6.0, soak and dissolve in a refrigerator at 4°C, overnight, centrifuge at 4500 rpm for 15 minutes, remove the precipitate, and collect snake venom supernatant;

[0037] (2), SP Sepharose Fast Flow cation exchange chromatography column (2.6cm×30cm) equilibrate at least 5 column volumes with PBS buffer solution of pH 6.0, put the above supernatant on the column, control the flow rate at 3.0ml / min, After the sample is finished, the column is equilibrated with 200ml buffer;

[0038] (3) Elute linearly with PBS buffer solution II containing 0.2M NaCl at pH 6.0, detect with an ultraviolet detector at 280 nm, collect activity peaks, and obtain a crude thrombin-like extract;

[0039] (4) The thrombin-like crude extract needs to be dialyzed at least th...

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Abstract

The invention discloses a method for extracting thrombin-like enzyme from Agkistrodon halys ussuriensis snake venom, which comprises the following steps of: soaking, dissolving and centrifuging snake venom; performing SP Sepharose Fast Flow cation exchange chromatography on supernate; dialyzing eluent containing active ingredients; and performing M-Aminothenyltrotionic Acid-Agarose affinity chromatography on the dialysis sample to obtain the thrombin-like enzyme with a single ingredient. The method can be operated at normal temperature, and has the characteristics of stable process, suitability for mass production, simple operation, short production period, high product purity and the like.

Description

technical field [0001] The invention relates to the technical field of medicine, and in particular provides a method for extracting a thrombin-like enzyme with hemostatic function from the venom of Agkistrodon halys. Background technique [0002] my country is very rich in snake venom resources. Among the more than 2,200 species of snakes in 10 families known in the world, there are as many as 175 species in 7 families in my country. my country is also the earliest country where snake venom was used for medical treatment. However, with the rapid development of biotechnology in foreign developed countries and the emphasis on natural substances, the research on the active ingredients of snake venom has been quite in-depth. In 1936, Klobusitzi and Koning first obtained partially purified thrombin-like enzyme from the venom of the American lancehead pit viper (B. jararaca). In 1967, Esnoff et al. isolated the venom-like thrombin from A.rhodostoma, Malaysia, and applied it to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
Inventor 薛百忠张妍王宏英薛雁李小军温丽丽王寒
Owner 辽宁远大诺康医药有限公司
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