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A method for isolating toxin protein from white jellyfish tentacles

A technology of white jellyfish and toxin protein, which is applied in the field of separation of toxin proteins caused by increased vascular permeability, can solve the problems of unfavorable separation and purification of impurity proteins, unstable and easy degradation of toxins, and difficulty in toxin collection, and achieves a beneficial effect on toxins. The effect of maintaining activity, being beneficial to separation and purification, and improving the purity of toxins

Active Publication Date: 2016-03-02
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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  • A method for isolating toxin protein from white jellyfish tentacles
  • A method for isolating toxin protein from white jellyfish tentacles
  • A method for isolating toxin protein from white jellyfish 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 pH 7.0 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) The crude poison obtained in step 1) was filtered through a microporous membrane with a pore size of 0.45 μm, and then separated by DEAESepharose FastFlow (2.6×10 cm), and eluted step by step, and the eluent was pH 7.0 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 membrane with a pore size of 0.45 μm, and then separate it on DEAESepharose FastFlow (2.6x10cm), and use step-by-step elution, and the eluent is: 15mmol / L, pH 7.1 Tris-HCl, flow rate 2.5ml / min, collect 900-1080ml eluate, concentrate to 2ml with 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 / L NaCl respectively.

[0042] 3) The concentrated solution obtained in step 2) is ...

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 FastFlow (2.6x10cm), and use step-by-step elution, and the eluent is: 20mmol / L, pH 7.2 Tris-HCl, flow rate 2ml / min, collect the eluate from 900-1080ml segment, and concentrate to 2ml with a 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.

[0048] 3) The concentrated so...

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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 rosy jellyfish (Cyaneanozakii 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 redness, swelling and stinging pain. Even allergic death, so in-depth study of jellyfish toxins has important practical significance for the prevention and treatment of jellyfish stings. 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 i...

Claims

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Application Information

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Patent Type & Authority Patents(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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