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Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method

A technology of cobra venom factor and neurotoxin, applied in the field of biomedicine, can solve the problems of unsuitable large-scale production process, ineffective utilization of snake venom protein, easy compression of chromatography column, etc., so as to reduce mutual interference, avoid protein denaturation, The effect of high comprehensive utilization

Inactive Publication Date: 2014-01-15
ORIENTOXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Because the content of CVF in the cobra venom is very average, it is difficult to extract, so there are few publicly reported CVF separation and purification methods. Bulletin, 1997Feb; 13(1): 85~87), using gel chromatography and then ion exchange chromatography for separation, gel chromatography using Bio-gel P200 column, ion exchange chromatography using DEAE cellulose DE 52 column, this method can prepare a small amount of samples, but the rigidity of the above two packing materials is poor, the chromatographic column is easy to compress, and takes a long time, it is not suitable for large-scale production process, and most snake venom proteins cannot be effectively utilized

Method used

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  • Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method
  • Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method

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Embodiment

[0032] In an embodiment, a combined separation and purification method of cobra venom factor and cobra venom neurotoxin adopts the following steps:

[0033] 1. Handling of sample crude virus

[0034] Weigh 20g of snake venom and dissolve it in 200ml of purified water or Tris buffer of pH5-8, centrifuge at 10000rpm at a temperature below 10°C for 10min, take the supernatant, and pass through a 0.22μm microporous membrane. The 5kDa ultrafiltration membrane is exchanged for the separation buffer;

[0035] 2. CM-Sepharose FF chromatography

[0036] The snake venom solution after the exchange buffer is loaded onto the CM Sepharose FF chromatographic column (5.0 × 30cm) equilibrated with Tris buffer solution of pH 7.5 and 20mmol / L with a peristaltic pump at a flow rate of 10ml / min, and eluted with the equilibrium buffer After the flow-through peak is collected, NaCl is added to the elution buffer to elute separately. The concentration of NaCl added is 0.1M, 0.2M, and 0.4M. The flo...

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Abstract

The invention discloses a cobra-venom factor and cobra-venom neurotoxin combined separation and purification method which includes the following steps: firstly the cobra-venom crude liquid is processed with centrifugation treatment at low temperature and high speed, and then small molecule substances, low molecular peptides and the like contained in the cobra-venom crude liquid are removed through a 3KDa ultrafiltration membrane. The two chromatography processes respectively adopt a CM-Sephrose. FF chromatographic column and a Superdex.200 gel chromatographic column; and cobra-venom neurotoxin and cobra-venom factor which are two active ingredients can be obtained in one production process. The method has the characteristics that the cobra-venom factor and the cobra-venom neurotoxin can be separated, the application efficiency of the cobra-venom is effectively improved, the purities of the products are improved and the method is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a combined separation and purification method of cobra venom factor and cobra venom neurotoxin. Background technique [0002] Cobra venom is a natural poisonous protein secreted by the venom gland of the cobra. Its chemical composition is very complex, containing a variety of proteins, polypeptides, enzymes and other small molecules, and has a wide range of biological activities. The main ones are neurotoxins, Cytotoxin, nerve growth factor and cobra venom factor (CVF), etc. With the development of modern biotechnology, many components of cobra venom have been isolated, purified and sequenced, and various components are widely used in theoretical research and clinical application of biochemistry, molecular biology, toxicology and pharmacology. [0003] Since Monaelesser and Taguet first reported the significant curative effect of cobra venom on the cases of pain ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/36C07K1/34C07K1/18C07K1/16C07K14/435
Inventor 吕宪峰刘志强王东林峰曹恒张坤
Owner ORIENTOXIN BIOTECH
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