A method for separating and purifying cobra venom neurotoxin in combination with ion exchange and hydrophobic chromatography

A technique for separation and purification of cobra venom, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of long separation and purification cycle and complicated operation steps, and achieve shortened cycle, simplified operation steps and high yield high effect

Active Publication Date: 2019-01-08
SUZHOU RENBEN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this method is owing to need to use the column chromatography methods such as gel column chromatography, ion exchange chromatography, hydrophobic chromatography and reverse phase chromatography in combination, not only operation steps are complicated, and separation and purification period is long (only the crude of king cobra venom It takes 576 hours to separate this step), so that its application range is limited to the laboratory

Method used

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  • A method for separating and purifying cobra venom neurotoxin in combination with ion exchange and hydrophobic chromatography
  • A method for separating and purifying cobra venom neurotoxin in combination with ion exchange and hydrophobic chromatography
  • A method for separating and purifying cobra venom neurotoxin in combination with ion exchange and hydrophobic chromatography

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Effect test

Embodiment 1

[0031] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0032] (1) Weigh 4.5g of crude cobra venom, add it to 0.045L water or 0.03M phosphate buffer with pH 6.0, stir to dissolve, centrifuge at 10000rpm at 4°C, discard the precipitate, and use 45μm PES to supernatant Microporous filter membrane is filtered, filters, and needs testing solution A, for subsequent use;

[0033] (2) Need testing solution A is purified by ion-exchange chromatography, the medium of ion-exchange chromatography is CM SepharoseFast Flow, with the 0.03M phosphate buffer of pH 6.0 as mobile phase A, to comprise 1M sodium chloride The 0.03M phosphate buffer with pH 6.0 is the mobile phase B. First, use 2 column bed volumes of mobile phase B for activation, then use 3 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution. The steps of gradient elution are: A:B volume ratio is 100%:0%, A:B volume rat...

Embodiment 2

[0037] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0038] (1) Weigh 4g of crude cobra venom, add it to 0.04L water or 0.01M phosphate buffer with pH 5.0, stir to dissolve, centrifuge at 8000rpm at 0°C, discard the precipitate, and use 45μm PES to micronize the supernatant. Pore ​​filter membrane is filtered, filters, and needs testing solution A, for subsequent use;

[0039](2) Purify need testing solution A by ion exchange chromatography, the medium of ion exchange chromatography is CM SepharoseFast Flow, with the 0.01M phosphate buffer of pH 5.0 as mobile phase A, to include 0.4M sodium sulfate The 0.01M phosphate buffer with pH 5.0 is used as mobile phase B. First, activate with 1 column bed volume of mobile phase B, then use 2 column bed volumes for equilibrium elution, and then gradient elution. The steps of gradient elution are: A:B volume ratio is 100%:0%, A:B volume ratio is 90%:10%, A:B v...

Embodiment 3

[0043] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:

[0044] (1) Weigh 5g of crude cobra venom, add it to 0.05L water or 0.05M phosphate buffer solution with pH 7.0, stir to dissolve, centrifuge at 12000rpm at 8°C, discard the precipitate, and use 45μm PES to micronize the supernatant. Pore ​​filter membrane is filtered, filters, and needs testing solution A, for subsequent use;

[0045] (2) Need testing solution A is purified by ion exchange chromatography, the medium of ion exchange chromatography is CM SepharoseFast Flow, with the 0.05M phosphate buffer of pH 7.0 as mobile phase A, to comprise 0.4M sodium phosphate The 0.05M phosphate buffer with pH 7.0 was used as the mobile phase B, firstly activated with 3 column bed volumes of mobile phase B, then used 4 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution, The steps of gradient elution are: A:B volume ratio ...

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Abstract

The invention particularly relates to a method for separating and purifying cobra-venom neurotoxin by combining ion exchange and hydrophobic chromatography. The method comprises the following steps: (1) weighing 4-5 parts by weight of cobra-venom crude product, adding the cobra-venom crude product into 0.04-0.05 part by volume of water or 0.01-0.05M of phosphate buffer solution with the pH of 5.0-7.0, stirring to dissolve the cobra-venom crude product, centrifuging the solution at the temperature of 0-8 DEG C, discarding precipitates, and filtrating supernatant so as to obtain a test sample solution A for later use; (2) purifying the test sample solution A through ion-exchange chromatography, carrying out gradient elution, carrying out HPLC (High-Performance Liquid Chromatography) detection, and carrying out collecting so as to obtain a test sample solution B for later use; and (3) purifying the test sample solution B through hydrophobic chromatography, carrying out gradient elution, carrying out HPLC detection, carrying out collecting, carrying out concentrating, and carrying out drying thereby obtaining a pure product of cobra-venom neurotoxin, wherein when the unit of parts by weight is g, the unit of parts by volume is correspondingly L. According to the method, operating steps are simplified, the separation and purification cycle is shortened, the yield is relatively high, the purity is relatively high, and thus the method is applicable to the industrial production of cobra-venom neurotoxin.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for separating and purifying cobra venom neurotoxin by combining ion exchange and hydrophobic chromatography. Background technique [0002] Cobra venom is the venom secreted by the venom glands of cobras. Its chemical composition is complex, containing a variety of proteins, polypeptides, enzymes and other small molecular substances, and has a wide range of biological activities. Since Monaelessert and Taguet first reported that cobra venom had a significant effect on pain caused by cancerous tissue compression of nerves in 1933, the analgesic effect of cobra venom has been studied in depth, and the cobra venom neurotoxin has shown a unique analgesic effect. Cobra venom neurotoxins can be divided into long chain and short chain. The short chain contains 60-62 amino acids and 4 disulfide bonds; the long chain contains 65-72 amino acids and 5 disulfide bonds. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C07K1/20C07K1/18
CPCC07K14/46
Inventor 秦正红李国庆
Owner SUZHOU RENBEN PHARMA
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