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Method for extracting toxin protein from white cyanea tentacles

A technology of white jellyfish and toxin protein, which is applied in the field of separation of toxin protein that causes increased vascular permeability, can solve the problems of unfavorable separation and purification of miscellaneous proteins, instability and easy degradation of toxin, and difficulty in toxin collection, so as to achieve beneficial toxin Maintain activity, facilitate separation and purification, and improve the effect of toxin purity

Active Publication Date: 2014-04-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, one of the difficulties in the study of jellyfish toxins is the difficulty in collecting toxins. Most of the toxins are extracted by crushing cnidocytes. The operation is cumbersome, time-consuming, the toxins are unstable and easy to degrade, and the introduction of a large amount of miscellaneous proteins is not conducive to further separation and purification.

Method used

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  • Method for extracting toxin protein from white cyanea tentacles
  • Method for extracting toxin protein from white cyanea tentacles
  • Method for extracting toxin protein from white cyanea tentacles

Examples

Experimental program
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Effect test

Embodiment 1

[0027] 1) Preparation of the crude poison of the white jellyfish: take 100g of the tentacles of the white jellyfish, put them in a -80°C ultra-low temperature refrigerator for 24 hours, add 400g of pH7.0 Tris-HCl buffer solution containing NaCl at 0-6°C, and the NaCl concentration is 0.1mol / L, pH 7.0 Tris-HCl concentration 10mmol / L, stirred until the tentacles melted, centrifuged at 8000g for 5min at 0-6°C, collected the supernatant buffer, concentrated to 5ml with a 3K ultrafiltration tube at 0-6°C.

[0028] 2) Filter the crude poison obtained in step 1) with a microporous filter membrane with a pore size of 0.45 μm, and then separate it on DEAESepharose Fast Flow (2.6x10cm), and use step-by-step elution, and the eluent is pH7. 0 Tris-HCl, the flow rate is 3ml / min, collect the eluate in the 900-1080ml section, and concentrate it to 2ml with a 3K ultrafiltration tube at 0-6°C.

[0029] The eluent was 10mmol / L, pH7.0 Tris-HCl containing 0, 0.1, 0.2, 0.5mol / L NaCl respectively....

Embodiment 2

[0039]1) Preparation of the crude poison of the white jellyfish: take 150g of the tentacles of the white jellyfish, put them in a -80°C ultra-low temperature refrigerator for 48 hours, add 300g of pH7.1 Tris-HCl buffer solution containing NaCl at 0-6°C, and the NaCl concentration is 0.15mol / L, stirred until the tentacles melted, centrifuged at 10000g for 10min at 0-6°C, collected the supernatant buffer, and concentrated to 5ml with a 5K ultrafiltration tube at 0-6°C.

[0040] 2) Filter the crude poison obtained in step 1) with a microporous filter membrane with a pore size of 0.45 μm, and then separate it on DEAESepharose Fast Flow (2.6x10cm), and use step-by-step elution, and the eluent is: respectively containing different NaCl concentrations 15mmol / L, pH7.1 Tris-HCl, flow rate 2.5ml / min, collect the eluate from 900-1080ml segment, and concentrate to 2ml with a 5K ultrafiltration tube at 0-6°C.

[0041] The eluent was 10mmol / L, pH7.1 Tris-HCl containing 0, 0.1, 0.2, 0.5mol / ...

Embodiment 3

[0045] 1) Preparation of the crude poison of the white jellyfish: take 200g of the tentacles of the white jellyfish, put them in a -80°C ultra-low temperature refrigerator for 72 hours, add 300g of pH7.2 Tris-HCl buffer solution containing NaCl at 0-6°C, and the NaCl concentration is 0.2mol / L, stirred until the tentacles melted, centrifuged at 10000g for 20min at 0-6°C, collected the supernatant buffer, and concentrated to 5ml with a 10K ultrafiltration tube at 0-6°C.

[0046] 2) Filter the crude poison obtained in step 1) with a microporous filter membrane with a pore size of 0.45 μm, and then separate it on DEAESepharose Fast Flow (2.6x10cm), and use step-by-step elution, and the eluent is: respectively containing different NaCl concentrations 20mmol / L, pH7.2 Tris-HCl, flow rate 2ml / min, collect 900-1080ml eluate, and concentrate to 2ml with 10K ultrafiltration tube at 0-6°C.

[0047] The eluent was 10mmol / L, pH7.2 Tris-HCl containing 0, 0.1, 0.2, 0.5mol / L NaCl respectively...

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Abstract

The invention relates to the technical field of biologics, in particular to a method for extracting a toxin protein which causes vascular permeability increase from white cyanea tentacles. The method comprises the following steps: unfreezing fresh white cyanea tentacles which are frozen at a super-low temperature at a low temperature, washing by a small quantity of buffer solution, collecting the buffer solution, centrifuging to obtain a supernatant and using the supernatant as cyanea crude toxin, and sequentially separating by an ion chromatographic column and a gel chromatographic column to obtain the toxin protein CnP45 which causes vascular permeability increase, wherein the molecular weight of the toxin protein is 45kD. The extracting process of the jellyfish toxin is simplified, the interference of impurity proteins is reduced, and an important method instruction is provided for the further separation and purification of jellyfish toxin ingredients.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating a toxin protein that causes increased vascular permeability from the tentacles of the white jellyfish Background technique [0002] White Xia jellyfish (Cyanea nozakii Kishinouye) is a common disastrous jellyfish in the coast of my country. It has a large number of stinging cells in its tentacles, which can release toxins after being stimulated. There are a large number of tourists every year. Fishermen are stung, and the victims have skin redness and swelling. pain, and even allergic death, so in-depth study of jellyfish toxins on the prevention and treatment of jellyfish stings has important practical significance. Jellyfish toxins are mainly protein substances, and their activities include enzyme activity, hemolytic activity, liver toxicity, cardiovascular activity, lethal activity, skin inflammatory activity and so on. At present, one of the difficulties in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/18C07K1/16
CPCC07K14/43595
Inventor 李鹏程薛伟于华华李荣锋邢荣娥刘松秦玉坤陈晓琳胡林峰
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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