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Agkistrodonacutus thrombin preparation method and uses

A technology of Haemocoagulase and Haemocoagulase, which is applied in the field of preparation of Haemocoagulase from Agkistrodon, can solve the problems of long production cycle, low yield, poor purity, etc., and achieve low production cost, high yield, good hemostatic effect

Active Publication Date: 2006-05-03
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

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Experimental program
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Effect test

Embodiment 1

[0046] 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 Then, gradient elution was carried out with 0.02-0.1M NaCl phosphate buffer at a flow rate of 20ml / min. The protein elution peak was 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.

[0047] The above collected Haemocoagulase from Agkistrodon haemocoagulase was identified by SDS-PAGE as consisting of two subunits, the molecular weight of A subunit was 1...

Embodiment 2

[0049] 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.

[0050] The above collected Haemocoagulase from Agkistrodon haemocoagulase was identified by SDS-PAGE electrophoresis as...

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PUM

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Abstract

The invention discloses a preparation method for Hemocoagulase of Agkistrodon acutus peculiar to China, which comprises dissolving, centrifuging at low temperature, dialyzing, DEAE-Sepharose FF chromatography, and Sephadex G25 chromatography. The product comprises A and B subunits with molecular weight as 15.0KD and 14.2KD respectively, and the amino acid sequences of first to twenty-five sites are DCSSGWSSYEGHCYKVFKQSKTWTA and DCPSDWSSYECHCYKPDEPKTWEDA respectively. This product can be used to haemostasis in clinic.

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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IPC IPC(8): C12N9/74A61K38/48A61P7/04
Inventor 王锡娟
Owner BEIJING KONRUNS PHARM CO LTD
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