Method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography, and preparation of cobra neurotoxin protein

A technology of ion exchange chromatography, separation and purification, applied in the field of separation and purification of cobra neurotoxin by double ion exchange chromatography, to achieve the effects of saving time and cost, scientific and reasonable process, and reducing losses

Active Publication Date: 2013-11-13
奔驰生物科技(云南)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for the separation and purification of cobotide, there are still problems of further improving the purity of cobotide and realizing the separation and purification of cobotide in industrialized production lines

Method used

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  • Method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography, and preparation of cobra neurotoxin protein
  • Method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography, and preparation of cobra neurotoxin protein
  • Method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography, and preparation of cobra neurotoxin protein

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Embodiment Construction

[0088] 1, prepare cobra neurotoxin, the technical parameters of embodiment 1-5 are shown in Table 3, and concrete steps are as follows:

[0089] (1) The first ion exchange chromatography separation and purification:

[0090] a) Dissolution of crude cobra venom:

[0091] Prepare 0.02M, adjust the pH5.6-pH6.8 phosphate buffer solution to be A solution;

[0092] Prepare liquid A containing 0.8M NaCl as liquid B;

[0093] Weigh 20-30 grams of crude cobra venom, dissolve it with 50-100 ml of solution A, centrifuge at 3000 rpm for 8-12 minutes, take the supernatant as snake venom, and set aside.

[0094]

[0095] The technical parameters of each specific embodiment of table 3

[0096]

[0097] b) Separation and purification on SP-Sephodex-C25 gel column:

[0098] Soak 600ml of SP-Sephodex-C25 gel in 500ml of 0.3NNaOH for 10 minutes, wash it with pure water until it is neutral, drain it, put it in a beaker, add 600ml of liquid A and pump it for 10 minutes, take it out and put...

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Abstract

The invention provides a method for separating and purifying cobra neurotoxin protein through dual-ion exchange chromatography. The method comprises the following steps of: (1) first separation and purification through ion exchange chromatography, namely, a) dissolution of crude cobra venom, and b) separation and purification through an SP-Sephodex-C25 gel column; and (2) second separation and purification through ion exchange chromatography, namely, a) balance of a SourceS(XK5030) column having a column volume of 500 ml by using 1000 ml of liquid A for future use; and b) SourceS(XK5030) column purification, and then collection of the cobra neurotoxin protein purified twice according to a standard purification chromatogram. The method provided by the invention is scientific and rational in process; and no organic reagent, no high-concentration salt and no protein modification method are used in production, so that the biological activity of cobratide can be maintained to an utmost extent. The method provided by the invention is advanced in process and simple to operate; the production period can be greatly shortened, the production time can be saved and the production cost can be reduced; and the method is completely suitable for an industrial production line.

Description

technical field [0001] The invention belongs to the field of separation and purification of biomedicine, and relates to a method and a preparation for separation and purification of cobra neurotoxin by double ion exchange chromatography. Background technique [0002] Cobotide is a cobra neurotoxin protein isolated and purified from the venom of Chinese cobra snakes. Cobotide mentioned in the present invention is the common name of cobra neurotoxin. [0003] Cobotide can play an analgesic effect by inhibiting the binding of acetylcholine to receptors at the neuromuscular junction postsynapse. Its biological activity directly affects the analgesic effect, including the intensity and duration of analgesia. [0004] Clinical application proves: Cobotide preparation has good analgesic effect, and the analgesic duration is long; side effects are small, and the safety factor is high; especially without the characteristics of drug tolerance and dependence, it is more and more acce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/18A61K38/17A61P29/00
Inventor 吕秋敏石聪博
Owner 奔驰生物科技(云南)有限公司
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