Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for separating and purifying cobra-venom neurotoxin by combining ion exchange and hydrophobic chromatography

An ion-exchange chromatography, separation and purification technology, applied in the field of biopharmaceuticals, can solve the problems of complicated operation steps and long separation and purification cycle, and achieve the effect of simplifying the operation steps, shortening the cycle and high yield

Active Publication Date: 2015-10-14
SUZHOU RENBEN PHARMA
View PDF2 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for separating and purifying cobra-venom neurotoxin by combining ion exchange and hydrophobic chromatography
  • Method for separating and purifying cobra-venom neurotoxin by combining ion exchange and hydrophobic chromatography
  • Method for separating and purifying cobra-venom neurotoxin by combining ion exchange and hydrophobic chromatography

Examples

Experimental program
Comparison scheme
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 Sepharose Fast Flow, with the 0.03M phosphate buffer of pH 6.0 as mobile phase A, to include 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 step of gradient elution is: A:B volume ratio is 100%:0%, A:B volume ratio...

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 Sepharose Fast 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 the mobile phase B, first activate with 1 column bed volume of mobile phase B, then use 2 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution , the step of gradient elution is: A:B volume ratio is 100%:0%, A:B volume ratio is 9...

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 Sepharose Fast Flow, with the 0.05M phosphate buffer of pH 7.0 as mobile phase A, to include 0.4M sodium phosphate The 0.05M phosphate buffer with a pH of 7.0 is the mobile phase B, first activate with 3 column bed volumes of mobile phase B, then use 4 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution , the step of gradient elution is: A:B volume ratio is 100%:0...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/20C07K1/18
CPCC07K14/46
Inventor 秦正红李国庆
Owner SUZHOU RENBEN PHARMA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com