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Agkistrodon acutus hemocoagulase-B

A technology of Agkistrodon akistrodon and hemagglutinase, applied in the field of serine protease, can solve the problem that blood clots cannot be dissolved by 5M urea

Active Publication Date: 2013-02-13
BEIJING KONRUNS PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of action of the two enzymes is different. Thrombin-like enzymes do not activate the ΧⅢ factor in the coagulation system, and their interaction with fibrinogen only produces non-cross-linked fibrin, which does not lead to thrombus formation, and the clot formed by it can be dissolved by 5M urea ;Thrombin can activate the ΧⅢ factor in the coagulation system, and it can interact with fibrinogen to form cross-linked fibrin, which can lead to thrombus formation, and the clot formed by it cannot be dissolved by 5M urea

Method used

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  • Agkistrodon acutus hemocoagulase-B
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  • Agkistrodon acutus hemocoagulase-B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Purification of snake venom hemagglutinin-B

[0033] Take 30g of Agkistrodon acutus snake venom freeze-dried powder (batch number: 20090701, Guangxi Snake Venom Research Institute), and dissolve it in a chromatography cabinet at 4°C with a volume of 10 times the weight of the snake venom and pre-cooled 0.01M pH7.4 PBS for 30 minutes. Centrifuge at 4°C and 10000g for 15 minutes, pour the centrifugal supernatant into a dialysis bag, add 10 times the weight of snake venom to the pre-cooled PBS to stir and suspend the centrifugal sediment, and centrifuge again. Combine the two centrifugal supernatants in a dialysis bag (with a molecular weight cut-off of 10,000D), and dialyze 0.01M pH7.4 PBS in a chromatography cabinet at 4°C for 24 hours, changing the fluid 3 times during the period. Load the pre-treated snake venom solution onto a DEAE-Sepharose Fast Flow anion exchange chromatography column pre-equilibrated with 0.01M pH7.4PBS, wash the column with 0.01M pH7.4PBS,...

Embodiment 2

[0036] Example 2 Purification of Snake Venom Hemagglutinin-B

[0037] Take 30 g of Agkistrodon acutus snake venom freeze-dried powder (batch number: 20090701, Guangxi Snake Venom Research Institute), and perform pretreatment according to the same method as in Example 1. The pretreated snake venom solution was subjected to the first DEAE-Sephrose FF column chromatography according to the same method as in Example 1, and the target substance appeared in the elution peak of 0.06M NaCl by enzyme activity determination and electrophoresis analysis, and the elution collection solution was combined. A total of 2000ml, using Millipore Pellicon 2 tangential flow ultrafilter (0.1M 2 Cut off10k membrane) ultrafiltration and concentration to 210ml, pour the 210ml ultrafiltration concentrate into a dialysis bag (10,000D), dialyze with 2000ml 0.01M pH7.4PBS at 4°C for 24 hours, changing the solution 3 times during the period. The enzyme solution after dialysis was loaded onto the DEAE-Sephrose...

Embodiment 3

[0038] Example 3 Serine protein properties test of Agkistrodon acutus hemagglutinin-B

[0039] The hemagglutinin-B isolated in Example 1 was diluted with physiological saline to an enzyme activity of 1 U / ml.

[0040] Prepare 1% bovine fibrinogen (Sigma) solution with physiological saline.

[0041] Dissolve phenylmethylsulfonyl fluoride (PMSF, Merck) with isopropanol, the solution concentration is 4mg / ml.

[0042] The experimental operation steps are as follows:

[0043] (1) Take 2ml of 1% bovine fibrinogen solution and keep it at 37°C for 5 minutes.

[0044] (2) Take three small test tubes, labeled 1#, 2#, 3#, and add 200μl of 1U / ml hemagglutinin-C solution to each tube.

[0045] (3) Add 10μl of distilled water to 1# test tube, 10μl of isopropanol to 2# test tube, and 10μl of PMSF to 3# test tube, and keep them in a 37°C water bath for 5 minutes.

[0046] (4) Perform separate agglutination test observations in the order of test tube numbers. Add 200 μl of 1% bovine fibrinogen solution at ...

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Abstract

The invention provides agkistrodon acutus hemocoagulase-B which is high-activity hemocoagulase separated from agkistrodon acutus venom. The hemocoagulase is single-stranded glycoprotein comprising 236 amino acids, and has an amino acid sequence shown as SEQ ID No. (sequence identification number) 1. The strand comprises 6 pairs of disulfide bonds; molecular weight of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is approximately 35kD; the molecular weight of the hemocoagulase after being deglycosylated is 26116.7Da; and an isoelectric point (pI) is 6.0. A hemocoagulase molecule is modified by heterozygotic polysaccharides at an Asn<77, 100, 229> (asparagine) site. The hemocoagulase is serine proteinase. The invention also provides a separation and purification method of the hemocoagulase, which comprises the steps that undissolved substances are removed through pretreating; then two times of anion-exchange column chromatography and one time of Sephdex-G75 molecular sieve chromatography are conducted; an active elution peak is acquired; dialysis and lyophilization are conducted; and the high-purity agkistrodon acutus venom hemocoagulase is obtained. The specific activity of the agkistrodon acutus venom hemocoagulase is not less than 2000U / mg; the analysis purity of HPLC (high performance liquid chromatography) can reach above 95%; and the yield is 0.25-0.30% calculated according to the weight of an agkistrodon acutus venom raw material.

Description

Technical field [0001] The present invention relates to a serine protease, specifically a Agkistrodon acutus venom hemagglutinin-B, and also relates to a separation and purification method thereof. Background technique [0002] There is a type of protease related to blood coagulation in the snake venom of Crotalinae, which is usually called "thrombin-like enzyme" (TLC). The apparent effects of thrombin-like enzymes and thrombin (thrombin) are similar, and both can convert fibrinogen in plasma into fibrin and "coagulate". However, the mechanism of action of the two enzymes is different. Thrombin-like enzymes do not activate factor XIII in the coagulation system, and their interaction with fibrinogen only produces non-crosslinked fibrin, which does not cause thrombosis. The clot formed by it can be dissolved by 5M urea. ; And thrombin can activate factor XIII in the coagulation system, which interacts with fibrinogen to form cross-linked fibrin, which can lead to thrombus formatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74A61K38/48A61P7/04
CPCA61K38/482A61P7/04C12N9/6418
Inventor 孙狄王锡娟
Owner BEIJING KONRUNS PHARM CO LTD
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