Snake venom protein component of ahylysantinfarctase at southern Anhui Province, extraction method and uses thereof

A technology of snake venom protein and venomous pit viper, which is applied in the extraction method and its application. and other problems to achieve the effect of improving immune system function and anti-tumor immune system function

Inactive Publication Date: 2009-02-18
芮景
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous experiments on the separation and purification of Agkistrodon halys venom, it was inevitable that a certain amount of thrombin-like enzymes would be mixed in the separated components. As a result, the anticoagulant component of the anticoagulant component often failed to Activity tends to be masked by thrombin-like blood clotting effects and less detected

Method used

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  • Snake venom protein component of ahylysantinfarctase at southern Anhui Province, extraction method and uses thereof
  • Snake venom protein component of ahylysantinfarctase at southern Anhui Province, extraction method and uses thereof
  • Snake venom protein component of ahylysantinfarctase at southern Anhui Province, extraction method and uses thereof

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Experimental program
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Effect test

Embodiment 1

[0033] The venom of Agkistrodon acutus in southern Anhui was purchased from Jinzhong Snake Farm in Ningguo City, Anhui Province.

[0034] CXG-1 column chromatography cabinet: contains TH-1000 gradient mixer, DHL-A computer constant flow pump, HD-5 computer ultraviolet detector, DBS-100 computer automatic partial collector (Shanghai Huxi Analytical Instruments) factory limited)

[0035] DEAE Sepharose Fast Flow gel, SP Sepharose Fast Flow gel, SephadexG75 gel are all produced by Swedish Pharmacia Company.

[0036] The BCA method protein content detection kit was produced by PIERCE Company of the United States.

[0037] a) Anion chromatography step: dissolve 1 gram of Agkistrodon venom crude poison in 0.01mol / LTris-Hcl solution with pH 8.0, centrifuge at 6000rpm, take 10ml of supernatant and put it on 200ml DEAE Sepharose FF column, use pH8 .0 0.01mol / L Tris-Hcl solution and pH 8.0 0.01mol / L Tris-Hcl solution containing 0.5mol / L NaCl for gradient elution, the temperature is 0℃...

Embodiment 2

[0050] a) Anion chromatography step: Dissolve 1.2 grams of crude venom of Agkistrodon venom in 0.008mol / LTris-Hcl solution with pH 7.8, centrifuge at 5000rpm, take about 10ml of the supernatant and put it on a 220ml DEAE Sepharose FF column , using 0.008mol / L Tris-Hcl solution with pH 7.8 and 0.008mol / L Tris-Hcl solution containing 0.45mol / L NaCl for gradient elution, the temperature is 0℃~10℃, the flow rate is 0.4ml / min, 20min Collect one tube, detect it at 280nm with an ultraviolet detector, record the chromatogram, collect the eluent from each peak according to the spectrum, measure the anticoagulant activity of each peak by APTT method, select the peak with the most obvious prolongation of anticoagulant time, and collect this The eluate from each tube of the peak was put into a dialysis bag, desalted and concentrated to obtain snake venom 1;

[0051] b) Cationic chromatography step: centrifuge 12ml of snake venom at 5000rpm, take 2ml of supernatant and put it on 200ml SP S...

Embodiment 3

[0054]a) Anion chromatography step: Dissolve 0.9 g of the crude venom of Agkistrodon acutina venom in a 0.012mol / LTris-Hcl solution at pH 8.1, centrifuge at 6500rpm, take 10ml of the supernatant and put it on a 180ml DEAE Sepharose FF column, Use 0.012 mol / L Tris-Hcl solution with pH 8.1 and 0.012 mol / L Tris-Hcl solution containing 0.55 mol / L NaCl for gradient elution, the temperature is 0°C to 10°C, the flow rate is 0.6ml / min, and the collection time is 20 minutes One tube, use a UV detector to detect at 280nm, record the chromatogram, collect the eluent from each peak according to the spectrum, measure the anticoagulant activity of each peak by APTT method, select the peak with the most obvious prolongation of anticoagulant time, and collect this peak The eluate from each tube was put into a dialysis bag, desalted and concentrated to obtain snake venom 1;

[0055] b) Cationic chromatography step: Centrifuge about 3ml of snake venom 1 at 6500rpm, take 3ml of the supernatant a...

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Abstract

The present invention relates to an agkistrodon acutus venom protein component and an extraction method and applications thereof. The agkistrodon acutus venom protein component is composed of venom proteins, the molecular weights of which are respectively 25KDa, 30KDa and 50KDa. The extraction method includes the following steps: (a) anion chromatography; (b) cation chromatography; (c) molecular sieve chromatography. Compared with the prior art, the extraction method utilizes the anion chromatography, the cation chromatography and the molecular sieve chromatography to extract a new anti-coagulating active component from agkistrodon acutus venom, and the prepared component has an anti-tumor effect and can improve the immune system.

Description

technical field [0001] The invention belongs to a snake venom protein component, an extraction method and an application thereof, in particular to a snake venom protein component, an extraction method and an application thereof. Background technique [0002] Snake venom is a natural poisonous protein secreted by snake venom glands, which contains a variety of proteins, enzymes, polypeptides and other small molecular substances, and has a wide range of biological activities. Ancient Chinese medical works such as "Shen Nong's Materia Medica" in the Western Han Dynasty, "Supplements to Materia Medica" in the Tang Dynasty, and "Compendium of Materia Medica" in the Ming Dynasty all have records of using snake venom to "fight poison with poison" to treat difficult and miscellaneous diseases. , treatment of rheumatic paralysis, numbness of limbs, malignant sores and other diseases. Modern medical research on the clinical effects of snake venom mainly focuses on the biological char...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/18C07K1/16A61K38/17A61P35/00A61P37/00
Inventor 芮景吉兆宁谢海棠张正明陈冬云张晔
Owner 芮景
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