High-purity anti-snake-venom serum and preparation method thereof

An anti-snake venom and high-purity technology, applied in the biological field, can solve the problems of long duration, high incidence, and serious illness, and achieve the effects of mild conditions, reduced protein content, and simple preparation methods

Active Publication Date: 2020-10-09
浙江毓昌生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antibody F(ab')2 content of this antiserum is about 90%, the effective F(ab')2 component that can neutralize the toxin only accounts for about 10% of the total F(ab')2 amount, and the rest F(ab')2 has no neutralizing effect on toxin
In addition, horse F(ab')

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  • High-purity anti-snake-venom serum and preparation method thereof
  • High-purity anti-snake-venom serum and preparation method thereof

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[0017] In a more specific embodiment of the present invention, a preparation method of high-purity antivenom comprises: a) cyanogen bromide-activated polysaccharide carrier, b) viper toxin coupled with cyanogen bromide-activated polysaccharide carrier, c) Purifying the antivenom F(ab')2 by using affinity chromatography.

[0018] Wherein, the viper toxin is selected from freshly taken viper toxins, viper toxins treated with ethylenediaminetetraacetic acid (EDTA), viper toxins treated with phenylmethylsulfonyl fluoride (PMSF) or viper toxins treated with ethylenediaminetetraacetic acid ( EDTA) and phenylmethylsulfonyl fluoride (PMSF) treated viper toxins or a mixture thereof.

[0019] In a preferred embodiment of the present invention, the specific steps of a preparation method of high-purity antivenom include:

[0020] a) Cyanogen bromide activated polysaccharide carrier: cyanogen bromide was dissolved in acetone and prepared to a concentration of 1M. Take the required amount ...

Embodiment 1

[0046] a) Preparation of cyanogen bromide activated agarose gel

[0047] 100 g of Sepharose 4B gel was washed with 60% acetone, suspended in 1000 ml of 60% acetone, and placed at -15°C. Add 14 ml of 1M cyanogen bromide (dissolved in acetone) to the Sepharose 4B suspension, stir rapidly under ice bath, and at the same time add dropwise 14 ml of triethylamine (TEA) solution (1.5M dissolved in 60% acetone). After 3 minutes, the entire reaction mixture was quickly poured into 1000 ml of washing solution (acetone:0.1N hydrochloric acid 1:1) pre-cooled in an ice bath. Stir well and store in a refrigerator at 2-8°C.

[0048] b) Five-step snake toxin coupling cyanogen bromide activated agarose gel preparation

[0049] The cyanogen bromide-activated agarose gel suspension was poured into a chromatographic column with a column height of 25 cm and a diameter of 5 cm. The gel was washed successively with ice pure water, pH 7.4, 50 mM phosphoric acid, and 0.15 M sodium chloride buffer so...

Embodiment 2

[0055] a) Preparation of cyanogen bromide activated agarose gel

[0056] 100 g of Sepharose 4B gel was washed with 60% acetone, suspended in 1000 ml of 60% acetone, and placed at -15°C. Add 14 ml of 1M cyanogen bromide (dissolved in acetone) to the Sepharose 4B suspension, stir rapidly under ice bath, and at the same time add dropwise 14 ml of triethylamine (TEA) solution (1.5M dissolved in 60% acetone). After 3 minutes, the entire reaction mixture was quickly poured into 1000 ml of washing solution (acetone:0.1N hydrochloric acid 1:1) pre-cooled in an ice bath. Stir well and store in a refrigerator at 2-8°C.

[0057] b) Preparation of phenylmethylsulfonyl fluoride-treated five-step snake toxin coupled with cyanogen bromide activated agarose gel

[0058] The cyanogen bromide-activated agarose gel suspension was poured into a chromatographic column with a column height of 25 cm and a diameter of 5 cm. The gel was washed successively with ice pure water, pH 7.4, 50 mM phosphor...

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Abstract

The invention discloses high-purity anti-snake-venom serum and a preparation method thereof. The high-purity anti-snake-venom serum is obtained mainly by the steps of activating a polysaccharide carrier by cyanogen bromide, coupling a toxic snake toxin to the cyanogen bromide activated polysaccharide carrier and purifying anti-snake-venom serum F (ab') 2 through an affinity chromatography method.The anti-snake-venom serum product with high neutralizing potency obtained by the preparation method has a good function of neutralizing snake venom, the amount of proteins without neutralizing toxiceffects in the product is greatly reduced, the occurrence rate of allergic reactions caused by heterogeneous proteins can be obviously reduced, and the severity of the allergic reactions can be reduced. In addition, the preparation method is simple, mild in condition and suitable for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-purity antivenom serum and a preparation method thereof. Background technique [0002] Viper, English Viper, refers to a snake that can secrete special venom. It is a cunning and vicious predator. Its keen senses make its prey have nowhere to escape, and its strange attacks make death follow. There are more than 40 species of venomous snakes in China, most of which are distributed in the vast provinces south of the Yangtze River. The known distribution areas include Anhui, Chongqing, Jiangxi, Zhejiang, Fujian, Hunan, Hubei, Guangxi, Guizhou, Guangdong and Taiwan. Among the venomous snakes, there are 10 most venomous snakes, including coral snakes, five-step snakes, king cobras, Zhoushan cobras, Agkistrodon chinensis, white-lipped bamboo leaf green, white-browed Agkistrodon, gray-blue flat-tailed sea snake, round-spotted viper, and golden ring snake. [0003] Snak...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/22
CPCC07K16/00C07K1/22
Inventor 方凯萍刘海昌朱复培杨乾敏时乐
Owner 浙江毓昌生物技术有限公司
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