Agkistrodon acutus hemocoagulase-B

A kind of technology of thorn pit viper and hemocoagulase, applied in the field of serine protease, can solve the problem that the blood clot cannot be dissolved by 5M urea, etc.

Active Publication Date: 2014-08-06
BEIJING KONRUNS PHARM CO LTD
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  • Summary
  • Abstract
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  • Claims
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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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Examples

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Embodiment 1

[0032] The purification of embodiment 1 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 chromatographic cabinet at 4°C for 30 minutes with pre-cooled 0.01M pH7.4 PBS in a volume 10 times the weight of the snake venom. Centrifuge at 4°C and 10,000g for 15 minutes, pour the centrifuged supernatant into a dialysis bag, and add pre-cooled PBS 10 times the volume of snake venom to the centrifuged pellet to stir and suspend, and centrifuge again. The supernatants from the two centrifuges were combined in a dialysis bag (molecular weight cut-off: 10,000D), and dialyzed against 0.01M PBS with pH 7.4 in a chromatographic cabinet at 4°C for 24 hours, during which the medium was changed 3 times. Load the pretreated snake venom solution onto the DEAE-Sepharose Fast Flow anion exchange chromatography column pre-balanced with 0.01M pH7.4PBS, wash the colu...

Embodiment 2

[0036] The purification of embodiment 2 snake venom hemagglutinin-B

[0037] Take 30 g of Agkistrodon acutus venom freeze-dried powder (batch number: 20090701, Guangxi Snake Venom Research Institute), and perform pretreatment according to the same method as in Example 1. Carry out the DEAE-Sephrose FF column chromatography for the first time by the snake venom solution of pretreatment by the same method of example 1, through enzymatic activity determination and electrophoresis analysis target object appears in the elution peak of 0.06MNaCl, combine elution collection liquid, A total of 2000ml was obtained, using a Millipore Pellicon 2 tangential flow ultrafilter (0.1M 2 cut off10k membrane) concentrated to 210ml by ultrafiltration, pour the 210ml ultrafiltration concentrate into a dialysis bag (10,000D), and dialyze with 2000ml 0.01M pH7.4PBS at 4°C for 24 hours, during which the solution was changed 3 times. The dialyzed enzyme solution was loaded onto a DEAE-Sephrose FF col...

Embodiment 3

[0038] Example 3 Serine protein property experiment of Haemocoagulase-B of Agkistrodon akistrodon

[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 saline.

[0041] Dissolve phenylmethylsulfonyl fluoride (PMSF, Merck) in isopropanol to a solution concentration of 4 mg / ml.

[0042] The experimental operation steps are as follows:

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

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

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

[0046] (4) Carry out the agglutination test separately according to the sequence of test tube numbe...

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Abstract

The present invention provides a Agkistrodon haemagglutinase-B, which is a highly active hemocoagulase isolated from Agkistrodon akistros venom, and the enzyme is a single-chain glycoprotein composed of 236 amino acids. It has the amino acid sequence shown in SEQ ID No.1. The chain contains 6 pairs of disulfide bonds; the SDS-PAGE molecular weight is about 35kD, and the molecular weight of the deglycosylated enzyme is 26116.7Da; the isoelectric point pI is 6.0. The enzyme molecule has hybrid polysaccharide modifications at Asn77, 100, and 229 positions. The enzyme is a serine protease. The present invention also provides a separation and purification method for the enzyme, which includes removing insoluble matter through pretreatment, and then passing through two anion exchange column chromatography and one Sephdex-G75 molecular sieve chromatography to collect the active elution peak, dialysis, freeze-drying, That is, high-purity snake venom hemagglutinin. Its specific activity is not less than 2000U / mg, its HPLC analysis purity can reach more than 95%, and its yield is 0.25%-0.30% based on the weight of snake venom raw materials.

Description

technical field [0001] The invention relates to a serine protease, specifically a haemagglutinase-B from Agkistrodon akistrodon venom, and also relates to a separation and purification method thereof. Background technique [0002] There is a protease related to blood coagulation in the snake venom of the subfamily Crotalinae, which is usually called "thrombin-like enzyme" (TLC for short). The apparent effect of thrombin-like enzymes and thrombin (thrombin) is 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 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 ; And thrombin can activate the ΧⅢ factor in the blood coagulation system, which can interact with fibrinogen to form cross-l...

Claims

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Application Information

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Patent Type & Authority Patents(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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