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Haemocoagulase acutus thrombin and preparation method thereof

The technology of thrush and thrombin, which is applied in the field of medicine and biology, can solve the problems of complex methods and low efficiency, and achieve the effects of protecting activity, improving efficiency and strong controllability.

Active Publication Date: 2016-05-18
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Most of the existing methods for extracting and purifying thrombin-like enzymes from Agkistrodon venom involve long-term dialysis and multiple liquid changes. In addition, the anion exchange chromatography step often requires linear elution or multi-step elution. There are problems such as complex methods and low efficiency

Method used

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  • Haemocoagulase acutus thrombin and preparation method thereof
  • Haemocoagulase acutus thrombin and preparation method thereof
  • Haemocoagulase acutus thrombin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Take 7.5g of Agkistrodon acutus venom powder, and dissolve it with 0.01M phosphate buffer solution (pH6.8) in a chromatographic cabinet at 4°C. company) for microfiltration (microfiltration column model: CFP-4-E-4MA, 0.45 μm, GE Healthcare), and the filtrate was collected and diluted with phosphate buffer. The above filtrate dilution was subjected to ultrafiltration (ultrafiltration column model: UFP-10-E-4MA, 10NMWC, GE Healthcare) using QuixStandandBenchtopSystem, and the sample solution was used for further chromatography. The chromatographic columns are all connected to the AKTAprimeplus protein separation and purification instrument (GE Healthcare).

[0032] The DEAE-Sepharose FastFlow chromatography column (GE Healthcare) was first equilibrated with 0.01M phosphate buffer solution (pH6.8), and then the above sample solution was loaded (10ml / min). After loading the sample, rinse (35ml / min) with 0.01M phosphate buffer solution (pH6.8) until no peak signal is detect...

Embodiment 2

[0042] Get Agkistrodon acutus snake venom powder 30g, stir and dissolve with 0.01M phosphate buffer saline solution (pH6.5) in 4 ℃ of chromatographic cabinets, after the snake venom dissolves completely, dilute with phosphate buffer saline, use QuixStandandBenchtopSystem (GE Healthcare company ) for microfiltration (microfiltration column model: CFP-4-E-4MA, 0.45 μm, GE Healthcare), collect the filtrate and add phosphate buffer for dilution. The above filtrate dilution was subjected to ultrafiltration (ultrafiltration column model: UFP-10-E-4MA, 10NMWC, GE Healthcare) using QuixStandandBenchtopSystem, and the sample solution was used for further chromatography. The chromatographic columns are all connected to the AKTAprimeplus protein separation and purification instrument (GE Healthcare).

[0043] The DEAE-Sepharose FastFlow chromatography column (GE Healthcare) was first equilibrated with 0.01M phosphate buffer solution (pH6.2), and then the above sample solution was loaded ...

Embodiment 3

[0048] Get Agkistrodon acutus snake venom powder 150g, stir and dissolve with 0.01M phosphate buffer saline solution (pH6.6) in 4 ℃ of chromatographic cabinets, after the snake venom dissolves completely, dilute with phosphate buffer saline, use Versarflux13 (GE Healthcare company ) for microfiltration (microfiltration column model: CFP-4-E-5A, 0.45 μm, GE Healthcare), collect the filtrate and add phosphate buffer for dilution. The above filtrate dilution was subjected to ultrafiltration using Versarflux13 (ultrafiltration column model: UFP-10-C-5A, 10NMWC, GE Healthcare), and the sample solution was used for further chromatography. The chromatographic columns were all connected to the AKTAprocess protein separation and purification instrument (GE Healthcare).

[0049] The DEAE-Sepharose FastFlow chromatography column (GE Healthcare) was first equilibrated with 0.01M phosphate buffer solution (pH 7.0), and then the above sample solution was loaded (400ml / min). After loading t...

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Abstract

The invention discloses a preparation method of high-activity haemocoagulase acutus thrombin. According to the method, a snake venom crude product is subjected to ultrafiltration through an ultrafiltration membrane, then subjected to anion column chromatography and eluted by a phosphoric acid buffer solution, heat source removal and desalination are conducted through a molecular sieve gel column, and finally the high-activity and high-purity thrombin is obtained. Compared with the prior art, the method has the following advantages that the method is suitable for large-scale production, operation is easy, efficiency is high, the yield of a prepared haemocoagulase acutus thrombin product is as high as 8%, the purity is as high as 99% or above, and the specific activity is not lower than 180 U / mg.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to a method for preparing thrombin from Agkistrodon akistrodon. Background technique [0002] Snake venom is a liquid with complex components secreted by the venom glands of venomous snakes. It contains a variety of proteins, polypeptides, enzymes and other small molecular substances, and has a wide range of biological activities. Many types of thrombin are found in the venom of Agkistrodon halys, Changbai Mountain white-browed viper and other snakes. [0003] Agkistrodonacutus is a species of Agkistrodonacutus in the family Viperidae, widely distributed in southern China. Haemocoagulase Acutus (Halase for short) is a thrombin-like enzyme isolated and purified from the venom of Haemocoagulase Acutus. It degrades fibrinogen into fibrin monomers by hydrolyzing the Aα chain of fibrin. Patent CN1218747 discloses the use of Agkistrodon akistrodon thrombin Hala...

Claims

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Application Information

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IPC IPC(8): C12N9/64
CPCC12N9/6418
Inventor 邓沁彭维王永刚吴忠
Owner SUN YAT SEN UNIV
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