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Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification

A technology of tetrodotoxin and chromatographic medium, applied in the field of separation and purification of biologically active molecules, can solve problems such as non-commercialization, achieve simple operation, reduce analysis cost and environmental pollution, and achieve good purification effect

Inactive Publication Date: 2012-04-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although immunoaffinity technology has been gradually applied in the field of separation and purification since the 1980s, the use of immunoaffinity chromatography to separate and purify tetrodotoxin has not been reported, and there is no commercial tetrodotoxin immunoaffinity chromatography (medium) column for sale

Method used

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  • Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification
  • Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification
  • Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification

Examples

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Effect test

Embodiment 1

[0025] Example 1: Preparation of tetrodotoxin immunoaffinity chromatography column.

[0026] Among them, tetrodotoxin antibody was purchased from Shanghai Yaji Biotechnology Co., Ltd., Sepharose CL-2B was purchased from Shanghai Ruigu Biotechnology Co., Ltd., and other required reagents were purchased from Guangdong Guanghua Technology Co., Ltd.

[0027] (1) Tetrodotoxin antibody and matrix pretreatment.

[0028] The commercially available tetrodotoxin antibody was dialyzed overnight in pH 6.5, 0.1 mol / L acetic acid-sodium acetate coupling buffer, and diluted to 0.1 mg / ml.

[0029] Weigh Sepharose CL-2B lyophilized powder activated by cyanogen bromide, fully swell in a container containing 1mmol / L HCL solution to obtain a certain volume of wet glue. Quickly wash twice with pH 6.5, 0.1 mol / L acetic acid-sodium acetate coupling buffer that is more than 8 times the volume of the wet gel, centrifuge at 5000r / min for 2 minutes, and dilute the wet gel to 20ml.

[0030] (2) Couplin...

Embodiment 2

[0038] Example 2: Purification of microbial source tetrodotoxin by immunoaffinity chromatography.

[0039] (1) Immunoaffinity chromatography column The affinity chromatography column prepared in Example 1 was used.

[0040](2) Sample pretreatment, puffer fish tissue source sample pretreatment: Puffer fish tissue (ovary, liver, etc.) is mashed, soaked and boiled in 0.1% acetic acid aqueous solution for 10 minutes, filtered to remove residue, extracted 3 times, combined with clarified liquid, concentrated by rotary evaporation The clarified liquid was suction-filtered through a 0.45um filter membrane to obtain a sample containing tetrodotoxin. Microbial source sample pretreatment: after centrifugation (8000×g, 30min) of the fermented broth of strains producing tetrodotoxin, collect the supernatant, adjust the pH to 4-5 with acetic acid, bathe in boiling water for 10 minutes, collect the supernatant after centrifugation, and rotate to evaporate Concentrate 10 times, and filter t...

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Abstract

The invention relates to a preparation method of an immunoaffinity chromatography medium and an application in tetraodotoxin purification. The preparation method of an immunoaffinity chromatography medium comprises the following steps of: activating a solid-phase carrier through a cyanogen bromide method; coupling a tetraodotoxin specific antibody to the swelled activated solid-phase carrier; sealing the immunoaffinity chromatography medium; and washing the immunoaffinity chromatography medium. The method for purifying tetraodotoxin by using the immunoaffinity chromatography medium comprises the following steps of: pretreating a sample containing tetraodotoxin; preparing a tetraodotoxin immunoaffinity chromatography column; loading the sample; eluting the column; and collecting the elution liquid. The immunoaffinity chromatography medium provided by the invention has high selectivity, strong retention and concentration capability on tetraodotoxin in the sample, water-phase operation, easiness in operation and good purification effect, the immunoaffinity chromatography column can be used repeatedly, a large amount of solvent can be saved, and analysis cost and environmental pollution are reduced.

Description

technical field [0001] The invention belongs to the field of separation and purification of bioactive molecules, and relates to a chromatographic material, in particular to an immunoaffinity chromatographic medium for the separation and purification of tetrodotoxin and its application. technical background [0002] Tetrodotoxin ( Tetrodotoxin, TIX ) is a highly toxic small molecule non-protein neurotoxin, which can bind highly specifically to sodium ion channel receptors in nerve cell membranes, thereby blocking sodium ion channels and inhibiting the conduction of nerve impulses. Therefore, tetrodotoxin has an excellent nerve analgesic effect, its anesthetic effect is more than 1000 times stronger than that of general anesthetics, and it has the advantage of not being addictive. It can replace chemical drugs and Chinese herbal medicines for drug rehabilitation. At the same time, it also has the effects of sedation, antiarrhythmia, prevention of renal failure and lowering bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/26B01J20/281B01J20/30B01D15/08C07K1/22
Inventor 余勃马春燕陆豫
Owner NANCHANG UNIV
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