Separation and purification procss of neurotoxin and cytotoxin of cobratoxin

A technology of cobra venom and neurotoxin, which is applied in the field of medicine and biology, can solve the problems of increasing the cost of drugs, hindering the popularization and use of drugs, and low extraction rate, and achieves the improvement of comprehensive utilization efficiency, comprehensive utilization efficiency and high extraction efficiency, and high salt content. low effect

Inactive Publication Date: 2006-03-01
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many domestic and foreign reports on the separation and purification of CNT and CTX (Chang, et al. Biochem Biophys Res Commun, 2002, 294 (3): 574; Lin, et al. J Protein Chem, 2002, 21 (2): 81; Li Fanzhu et al., Chinese Pharmacist, 2004, 7(9): 659; Zhang Mingfang et al., Journal of Fujian Medical University, 2004, 38(1): 1), also have relevant patented technology (WO 01 / 03710 and CN 1102570A), but both Different media such as SP-Sephadex, CM-Sephadex, Sephadex and Mono Q are used for separation and purification of ion exchange chromatography and molecular sieve chromatography. Although a single component with high purity can be obtained, the extraction rate is low and expensive Protein purification equipment increases the cost of drugs and hinders the further promotion and use of such drugs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Take 2.5 g of Zhejiang cobra snake venom stock solution and 25 ml of analytically pure acetone. Put the stock solution of snake venom in a mortar, add 5ml of acetone to grind it into a homogenate, filter the homogenate with filter paper, wash with the remaining 20ml of acetone, leave it at room temperature to volatilize the acetone, and obtain the snake venom acetone powder.

[0023] (2) Dissolve the snake venom acetone powder in 25ml of distilled water, adjust the pH to 4.5 with 50% acetic acid solution, centrifuge at 10000rpm for 5min (4°C), take the supernatant, and record the volume.

[0024] (3) According to the supernatant volume, add 25% polyacrylic acid (polyacrylic acid, PAA) to a final concentration of 4%, record the amount of PAA added, place it at 4°C for 40min, centrifuge at 2000rpm for 3min (4°C), take the precipitate and dissolve it in In appropriate amount of distilled water, adjust the pH to 9.7 with 0.5mol / L sodium carbonate solution, add sodium ch...

Embodiment 2

[0027] (1) Take 4.0 g of cobra snake venom stock solution produced in Zhejiang, and 40 ml of acetone of analytical grade. Put the stock solution of snake venom in a mortar, add 7ml of acetone to grind it into a homogenate, filter the homogenate with filter paper, wash with the remaining 33ml of acetone, leave it at room temperature to volatilize the acetone, and obtain the snake venom acetone powder.

[0028] (2) Dissolve the snake venom acetone powder in 40ml of distilled water, adjust the pH to 4.0 with 50% acetic acid solution, centrifuge at 10000rpm for 8min (4°C), and take the supernatant.

[0029] (3) Same as step (3) of Example 1, but the final concentration of PAA is 5%.

[0030] (4) The supernatant was separated with a Sephadex G-50 (ultrafine particle) gel column, and eluted with 8mmol / L phosphate buffer (pH7.5, containing 0.3mol / L sodium chloride). The main peak of elution is neurotoxin, and the second main peak of elution is cytotoxin, and the solution of each fra...

Embodiment 3

[0032] (1) Take 4.0 g of cobra snake venom stock solution produced in Zhejiang, and 40 ml of acetone of analytical grade. Put the stock solution of snake venom in a mortar, add 7ml of acetone to grind it into a homogenate, filter the homogenate with filter paper, wash with the remaining 33ml of acetone, leave it at room temperature to volatilize the acetone, and obtain the snake venom acetone powder.

[0033] (2) Dissolve the snake venom acetone powder in 40 ml of distilled water, adjust the pH to 5.0 with 50% acetic acid solution, centrifuge at 10000 rpm for 10 min (4° C.), and take the supernatant.

[0034] (3) Same as step (3) of Example 1, but the final concentration of PAA is 3.2-3.5%.

[0035](4) The supernatant was separated with a Sephadex G-50 (ultrafine particle) gel column, and eluted with 10mmol / L phosphate buffer (pH7.5, containing 0.3mol / L sodium chloride). The main peak of elution is neurotoxin, and the second main peak of elution is cytotoxin, and the solution...

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Abstract

The present invention discloses separation and purification process of neurotoxin and cytotoxin of cobratoxin, and the process can separate neurotoxin and cytotoxin of cobratoxin simultaneously and raise the comprehensive utilization efficiency of cobratoxin. The process includes first treating cobratoxin liquid with acetone to eliminate liposoluble components from cobratoxin liquid, regulating the pH value of the water solution of cobratoxin and acetone powder with acid, centrifuging to eliminate the acid protein and polypeptide, specifically PAA adsorbing and precipitating the alkali protein and alkali polypeptide in cobratoxin liquid, centrifuging to separate PAA-alkali protein/polypeptide composition and eliminate water soluble components; desorbing PAA-alkali protein/polypeptide with pH variation, and eliminating PAA with calcium chloride; and separating and purifying neurotoxin and cytotoxin of cobratoxin with Sephadex G-50. The product has low salt content and may be frozen directly.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to a separation and purification method of cobra venom neurotoxin and cytotoxin. Background technique [0002] Cobra venom is a natural poisonous protein secreted by the venom glands of cobras. 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. 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 cancerous tissue oppressed nerves and caused pain in 1933, the analgesic effect of cobra venom has been deeply studied, and cobra venom neuro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/14
Inventor 童富淡张海花金苏华
Owner ZHEJIANG UNIV
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