Agkistrodonacutus thrombin preparation method and uses

A technology for hemocoagulase from Viper serrata, which is applied in the field of preparation of Viper serrata hemocoagulase, can solve the problems of long production cycle, low yield, poor purity and the like, and achieves low production cost, high yield and good quality. The effect of hemostatic action

Inactive Publication Date: 2009-08-05
BEIJING KONRUNS PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Agkistrodonacutus thrombin preparation method and uses
  • Agkistrodonacutus thrombin preparation method and uses
  • Agkistrodonacutus thrombin preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] After dissolving 10g of Agkistrodon halys venom with phosphate buffer, centrifuge at 5000rpm for 30min, and then dialyze the centrifuged supernatant in a dialysis bag for 24 hours, then go to DEAE-Sepharose column chromatography, first use 0.02M NaCl phosphate buffer elution, and then gradient elution with 0.02-0.1M NaCl phosphate buffer at a flow rate of 20ml / min, the protein elution peaks were collected, and then put into a dialysis bag for dialysis for 24 hours. The dialyzed solution was collected on DEAE-Sepharose chromatography column, and after gradient elution with 0.04, 0.06, 0.08 and 1.0M NaCl phosphate buffer, it was activated by coagulation of human plasma, identified by HPLC and SOS-PAGE, and determined that 0.08M The NaCl elution fraction (volume 200ml) was Agkistrodon haemagglutinin.

[0048] The above collected Haemocoagulase from Agkistrodon haematoblasts was identified by SDS-PAGE as consisting of two subunits, the molecular weight of subunit A was 15.0...

Embodiment 2

[0050] After dissolving 15g Agkistrodon halys venom with phosphate buffer solution, centrifuge at 5000rpm for 30min, and then dialyze the centrifuged supernatant in a dialysis bag for 24 hours, then go to DEAE-Sepharose chromatography column chromatography, in 0.02M NaCl phosphate buffer solution Elution, and then gradient elution with 0.02-0.1M NaCl phosphate buffer at a flow rate of 20ml / min, the protein elution peaks were collected, and then put into a dialysis bag for dialysis for 24 hours. The dialyzed solution was collected and then subjected to DEAE-Sepharose chromatography column chromatography. After gradient elution with 0.04, 0.06, 0.08 and 1.0M NaCl phosphate buffer, it was activated by coagulation of human plasma, identified by HPLC and SOS-PAGE, and determined that 0.08M The NaCl elution fraction (volume 230 ml) was Agkistrodon haemagglutinin.

[0051] The above collected Haemocoagulase from Agkistrodon haemocoagulase was identified by SDS-PAGE electrophoresis as...

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Abstract

The invention discloses a preparation method and application of haemocoagulase of Agkistrodon akistrodon. It comprises steps of dissolving snake venom, low-temperature centrifugation, dialysis, DEAE-Sepharose FF chromatography, dialysis, DEAE-Sepharose FF chromatography and Sephadex G25 chromatography. It is isolated and purified from the venom of Agkistrodon akistrodon, which is unique to China. It is composed of two subunits, A and B, and has the effect of promoting blood coagulation and hemostasis. The molecular weights of the two subunits A and B are 15.0KD and 14.2KD. The amino acid sequence from the first to the 25th position of the N-terminal of the A subunit is DCSSGWSSYEGHCYKVFKQSKTWTA, and the amino acid sequence from the first to the 25th position of the N-terminal of the B subunit is DCPSDWSSYECHCYKPDEPKTWEDA. Agkistrodon haemocoagulase can be used for clinical hemostasis of various types of bleeding in humans.

Description

technical field [0001] The invention relates to a preparation method and application of haemocoagulase of Agkistrodon akistrodon. In particular, it refers to a preparation method of a snake venom-like thrombin with hemostatic effect isolated and purified from the venom of Agkistrodon akistros, a special product in China. Background technique [0002] Snake venom thrombin (Thrombin-like enzymes, TLEs) is a class of proteases with thrombin-like activity isolated and purified from snake venom, which can directly and specifically hydrolyze fibrinogen to form fibrin and promote blood coagulation. The thrombin-like preparations for hemostasis that have been approved for production abroad and domestically are mainly Lizhizhi, which is a thrombin-like agent purified from the venom of Bothrops Atrox. The hemostatic drug named "Reptilase" developed by Switzerland's Basel Co., Ltd. is a thrombin-like enzyme extracted from the venom of the unique Brazilian cave viper. my country import...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74A61K38/48A61P7/04
Inventor 王锡娟
Owner BEIJING KONRUNS PHARM CO LTD
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