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Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin

A technology of toxin protein and nematocyst, which is applied in the field of separation and purification of lethal toxin protein, can solve the problems of separation and purification of jellyfish toxin, difficulty in separation and purification of jellyfish toxin, troublesome separation and purification of jellyfish toxin, etc., and shorten the separation time , high yield and less separation steps

Active Publication Date: 2013-07-03
水母娘娘海洋生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] But, so far the research of jellyfish toxin mainly concentrates on jellyfish crude venom, and its main reasons have the following aspects: first, jellyfish crude toxin contains many kinds of protein components, and the properties between some of them ( Such as: molecular weight, charged number, hydrophobicity, etc.) are very similar, which brings a lot of trouble to the separation and purification of jellyfish toxin; The influence of environmental factors leads to the reduction or even loss of its activity, which makes it difficult to maintain its activity during the separation and purification process
Third, different types of jellyfish have great differences in their physiological structure, toxin composition and activity contained in the body, which makes it difficult to directly imitate the separation and purification of different types of jellyfish toxins, and can only be obtained through complicated experiments. get
Therefore, the separation and purification of jellyfish toxins is very difficult

Method used

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  • Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin
  • Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin
  • Method for separating lethal toxin protein from cyanea nozakii nematocyst toxin

Examples

Experimental program
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Embodiment 1

[0024] 1) Preparation of nematocyst cells from jellyfish toxin: 1 kg of frozen jellyfish tentacles were autolyzed at 2°C overnight, and the tentacle fragments were removed by 20-mesh standard sampling sieve. Centrifuge at a high speed for 20 min at ℃ and collect the lower precipitate, and use 0.154mol·L -1 The NaCl solution was washed repeatedly with physiological saline at 2°C for 3 times until the supernatant was clear, and the nematocyst cell pellet was freeze-dried and stored at -20°C for future use.

[0025] 2) Extraction of jellyfish toxin: take step 1) 0.5g of nematocyst cells of jellyfish and 20mL pH7.810mM Tris-HCl buffer pre-cooled at 2°C in a beaker, and use ultrasonic power of During crushing at 200kw for 30 minutes, the work / interval is 5s / 30s. After crushing, centrifuge at 10,000g for 20min at 4°C, the supernatant is the nematocyst cytotoxin of jellyfish, and use the Coomassie brilliant blue method to measure nematocyst cells in the supernatant with bovine serum...

Embodiment 2

[0035] 1) Preparation of nematocyst cells from jellyfish toxin: After autolyzing 2kg of frozen jellyfish tentacles at 4°C overnight, use a 40-mesh standard sample sieve to remove tentacle fragments, then take the supernatant and dry it in 12000g, 4 Centrifuge at high speed for 10 min at ℃ and collect the lower precipitate, and use 0.154mol·L -1 The NaCl solution was washed repeatedly with physiological saline at 4°C for 3 times until the supernatant was clear, and the nematocyst cell pellet was freeze-dried and stored at -20°C for future use.

[0036] 2) Extraction of jellyfish toxin: take step 1) 1.0g of jellyfish nematocyst cells and 30mL pH7.820mM Tris-HCl buffer pre-cooled at 4°C in a beaker, and use ultrasonic power of During the crushing at 300kw for 30 minutes, the work / interval is 5s / 30s. After crushing, centrifuge at 12000g for 10min at 4°C, the supernatant is the nematocyst cytotoxin of jellyfish, and use the coomassie brilliant blue method to measure nematocyst cel...

Embodiment 3

[0046] 1) Preparation of nematocyst cells from jellyfish toxin: after autolyzing 3 kg of frozen jellyfish tentacles at 6°C overnight, use a 60-mesh standard sampling sieve to remove tentacles fragments, then take the supernatant and dry it in 15000g, 6 Centrifuge at a high speed for 5 min at ℃ and collect the lower precipitate, and use 0.154 mol·L -1 The NaCl solution was washed repeatedly with physiological saline at 6°C for 3 times until the supernatant was clarified, and the nematocyst cell pellet was freeze-dried and stored at -20°C for future use.

[0047] 2) Extraction of jellyfish toxin: take step 1) 2.0g of jellyfish nematocyst cells and 50mL pH7.830mM Tris-HCl buffer precooled at 6°C are placed in a beaker, and the ultrasonic power is During 60 minutes of crushing at 400kw, the work / interval is 5s / 30s. After crushing, centrifuge at 15000g for 5min at 6°C, the supernatant is the nematocyst cytotoxin of jellyfish, and use coomassie brilliant blue method, with bovine se...

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Abstract

The invention relates to the technical field of marine organisms, in particular to a method for separating and purifying a cyanea nozakii lethal toxin protein. The method comprises the following steps of: ultrasonically crushing cyanea nozakii nematocyst cells to extract a jellyfish crude toxin, dialyzing and desalting, and separating and purifying by affinity chromatographic separation and an anion exchange chromatographic technology in turn to obtain a jellyfish toxin protein with lethal activity, wherein through measurement, the molecular weight of the toxin protein is 30kDa, and the lethal dose 50 (LD50) of the toxin protein to 1g of zebra fish is 0.56mu g. The method has the characteristics that: the method is quick, simple and convenient and provides important guidance for obtainingthe cyanea nozakii lethal toxin protein.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for separating and purifying lethal toxin protein from Cyanea nozakii nematocyst cytotoxin. Background technique [0002] Jellyfish (jellyfish) is a kind of colloidal zooplankton, mainly including four groups: hydromedus, siphonophores, pot jellies and comb jellies of cnidaria. There are many kinds of jellyfish and they are widely distributed. There are about 1,000 species of jellyfish recorded in the world, of which about 400 species are known in my country's sea areas. my country is one of the countries with a very long coastline, from north to south across the cold temperate zone, temperate zone, subtropical zone and tropical zone, and there are a large number of jellyfish distributed in almost all coastal areas of our country, among which the number of jellyfish is particularly abundant. In recent years, frequent outbreaks of jellyfish have caused many swimmers to be stung. In mild ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435
Inventor 李鹏程李荣锋于华华冯金华邢荣娥刘松秦玉坤李克成孟祥涛崔金会
Owner 水母娘娘海洋生物科技有限公司
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