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Preparation method of jellyfish cardiovascular toxin crude extract

A cardiovascular and crude extract technology, which is applied in the field of preparation of jellyfish cardiovascular toxin crude extract, can solve the problem that it is difficult to meet the needs of toxin group separation and purification, and achieve the effect of facilitating purification, less introduction and easy operation

Active Publication Date: 2014-03-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, these two methods for the preparation of crude extracts of jellyfish toxins have limitations, and it is difficult to meet the needs of subsequent separation and purification of toxins

Method used

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  • Preparation method of jellyfish cardiovascular toxin crude extract
  • Preparation method of jellyfish cardiovascular toxin crude extract
  • Preparation method of jellyfish cardiovascular toxin crude extract

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Method of the present invention (referred to as quick method) prepares jellyfish cardiovascular toxin crude extract

[0024] PBS preparation: weigh 8g NaCl, 0.2g KCl, 3.63g Na 2 HPO 4 ·6H 2 O and 0.24gKH 2 PO 4 , dissolved in 900mL distilled water, adjusted the pH value to 7.4 with hydrochloric acid, added distilled water to make up to 1L, and finally filtered through a microporous membrane with a pore size of 0.20 μm, and pre-cooled at 4°C.

[0025] 1. Collection of jellyfish: Cyanea capillata was collected from Shengsi County, Zhoushan City, Zhejiang Province. Select jellyfish individuals with good vitality and well-developed tentacles, and use hand-made nets to catch them. First, put the captured jellyfish into a water tank filled with seawater and let it stand for 10 minutes until it returns to its natural activity state.

[0026] 2. Separation of tentacles: Remove impurities from tentacles, carefully distinguish different tissues such as tentacles and mouth-w...

Embodiment 2

[0031] Preparation of jellyfish toxin crude extract by autolysis method in prior art

[0032] Faxingxia jellyfish is collected from Shengsi County, Zhoushan City, Zhejiang Province. Take 100g of the wrist tissue (a mixture of tentacles and wrists), add an equal volume of 3.34% artificial seawater 100mL, autolyze for 4 days, stir twice a day with a magnetic stirrer, 10min each time; 100-mesh cell sieve Filtrate 3 times, centrifuge the filtrate 10000×g 3 times, 10min each time, collect the supernatant; place the supernatant in a dialysis bag with a molecular weight cut-off of 1000Da, dialyze in pre-cooled PBS at 4°C for 8h, then dialyze at 4°C Centrifuge at 10,000×g for 10 minutes, and the supernatant is the jellyfish toxin crude extract (TE, tentacle extract). (Artificial seawater preparation: weigh 28g of NaCl, MgCl 2 ·6H 2 o 5 g, KCl0.8g, CaCl 2 1.033g, add distilled water to 1L, mix well, filter through a microporous membrane with a pore size of 0.20μm, and pre-cool at ...

Embodiment 3

[0034] Comparison of crude extracts of jellyfish toxins prepared by autolysis method and rapid method

[0035] 1. Comparison of protein purity

[0036] Carry out SDS-PAGE electrophoresis with the toxin crude extract and the protein molecular weight standard reference substance (Marker) prepared by the quick preparation method and the autolysis method, using 5% stacking gel and 12% separating gel respectively, and loading 20 μl of the sample on a single well (total protein 20μg), stacking gel and separating gel voltage were 80v and 160v respectively, after the electrophoresis was finished, it was stained with silver staining.

[0037] The result is as figure 1 As shown, compared with the autolysis method, the content of large molecular weight tissue protein (non-toxin protein) in the jellyfish cardiovascular toxin crude extract produced by the rapid method is significantly reduced, suggesting that the method of the present invention can effectively reduce the introduction of n...

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Abstract

The invention relates to the technical field of marine organisms and aims at providing a preparation method of a jellyfish cardiovascular toxin crude extract. The method is a method of crudely extracting jellyfish cardiovascular toxin by taking fresh jellyfish tentacles (not oral arms or a whole oral arm tissue) as materials, and stimulating nematocysts by short-time fierce vibration by utilizing a physical stimulation method, so that most of the nematocysts are emitted in a concentrated manner to discharge venom in the nematocysts; then, centrifuging and collecting supernatant. The method disclosed by the invention is simple and convenient to operate, consumed time is shortened by 2-3 days in comparison with the prior art, and non-toxin impurity proteins are introduced less; moreover, the crude extract does not contain hematoxin and is beneficial to further purifying a cardiovascular component.

Description

technical field [0001] The invention relates to the technical field of marine organisms, in particular to a method for preparing jellyfish cardiovascular toxin crude extract. Background technique [0002] Jellyfish stings have become the most common marine injury, and jellyfish venom is the main cause of the series of symptoms after stings. Jellyfish toxin has multiple biological activities such as cardiovascular, hemolysis, nerve, liver, skin and muscle, and protease. It is a class of marine peptide toxins with high research value. [0003] The circulatory function damage caused by cardiovascular toxins is the main cause of death from jellyfish stings, but the components of cardiovascular toxins are less abundant in jellyfish toxins. The previous methods for the preparation of crude extracts of jellyfish toxins have shortcomings such as time-consuming, easy introduction of non-toxin components, and small preparation volumes. In particular, the interference of other high-ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/34
CPCC07K14/43595
Inventor 张黎明郑杰民陆佳阮增良成熙王蓓蕾张博刘丹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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