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Preparation method and application of veterinary-use A type clostridium perfringens toxins

A technology of Clostridium perfringens and perfringens, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of vaccine production waste, manpower, and time-consuming

Active Publication Date: 2019-06-28
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vaccines used in the market generally use the enzymatic digestion solution of beef and liver as raw materials to prepare the culture medium. The preparation process of the culture medium by this method is cumbersome, time-consuming, and requires a lot of manpower. Insufficient toxin production performance is unstable, which affects the quality of the vaccine, and also brings great waste and high cost to vaccine production

Method used

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  • Preparation method and application of veterinary-use A type clostridium perfringens toxins
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  • Preparation method and application of veterinary-use A type clostridium perfringens toxins

Examples

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Effect test

Embodiment 1

[0111] Embodiment 1, the cultivation of type A clostridium perfringens and the preparation of type A clostridium perfringens toxin

[0112] One, the cultivation of type A clostridium perfringens of the present invention and the preparation of type A clostridium perfringens toxin

[0113] The present embodiment relates to strain culture medium and fermentation medium as follows:

[0114] The solvent of the culture medium is purified water; the solute and concentration are: 30g / L casein peptone, 15g / L yeast extract powder, 10mM Na 2 CO 3 , 40mM Na 2 HPO 4 12H 2 O, 10mM KH 2 PO 4 , pH 7.5. Pack into small tubes and autoclave at 110°C for 30 minutes.

[0115] The solvent of the fermentation medium is purified water; the solute and concentration are: 30g / L casein peptone, 15g / L yeast extract powder, 0.1g / L CaCl 2 , 0.0014g / L ZnSO 4 ·7H 2 O, 5g / L Na 2 HPO 4 12H 2 O, 0.5g / L KH 2 PO 4 , 10g / L glucose, pH 7.5. Autoclave at 116°C for 30min.

[0116] The preparation meth...

Embodiment 2

[0136] Embodiment 2, preparation of type A Clostridium perfringens toxoid and type A Clostridium perfringens toxoid vaccine

[0137] 1. Inactivation of Clostridium perfringens type A and detoxification of toxin

[0138] Traditional inactivation method: add formaldehyde solution (40%) to the fermentation product obtained in Step 1 of Example 1 at a volume fraction of 0.5%, inactivate and detoxify at 37°C for 7 days, and obtain the inactivated and detoxified bacterial liquid .

[0139] Improved inactivation method: add L-lysine to the fermented product obtained in Step 1 of Example 1, so that the mass fraction in the fermented product is 0.7%, dissolve and mix well, and then add L-lysine at a ratio of 0.5% by volume Add formaldehyde aqueous solution (40%), adjust the pH value to 6.8 with 10M sodium hydroxide, inactivate and detoxify at 37°C for 7 days, and obtain the inactivated and detoxified bacterial liquid.

[0140] 2. Detection of Clostridium perfringens type A toxin inac...

Embodiment 3

[0149] Embodiment 3, the immune effect detection of type A Clostridium perfringens toxoid vaccine on rabbits

[0150] The A-type Clostridium perfringens toxoid vaccine prepared in Example 2, traditional craft vaccine (traditional craft vaccine is the vaccine prepared by "Chinese Veterinary Biological Products Regulations" 2000 edition 101-102 pages), commodity The vaccine (rabbit Clostridium perfringens (type A) inactivated vaccine, Shandong Huahong Bioengineering Co., Ltd., 201801001 batch) was injected subcutaneously into each 4 rabbits on the back of the neck, and different doses were immunized respectively (as shown in Table 6). (shown), and 2 unimmunized rabbits were set as controls at the same time, and the second immunization was carried out 21 days after immunization (the immunization dose and inoculation method were the same as the first immunization). Before immunization, 21 days and 35 days after the first immunization, 5 mL of blood was collected from the middle ea...

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Abstract

The invention discloses a preparation method and application of veterinary-use A type clostridium perfringens toxoids. The preparation method of the toxoids includes the following steps that A type clostridium perfringens production strains are inoculated into a culture medium for fermentation and culture, a fermentation product is obtained, then the fermentation product is inactivated and detoxified in L-lysine and a formaldehyde aqueous solution under the pH of 6.8, and centrifugal supernatant of the successfully inactivated and detoxified qualified bacterial solution refer to toxoids. The culture medium includes casein peptone, yeast extract powder, CaCl2, ZnSO4 7H2O, Na2HPO4 12H2O, KH2PO4, and glucose. The toxicity of the A type clostridium perfringens toxoids prepared by the method can be raised to be 12.5 times of the standard for seedling cultivation, the neutralization titer, in first and second immune serum of domestic rabbits, of the toxoids can be raised to 4 and 50 times ofa traditional process respectively and maximally, and the ratio of the serum titer to cost can be raised to 48.8 and 1216.4 times of the traditional process.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a preparation method and application of veterinary Clostridium perfringens type A toxoid. Background technique [0002] Clostridium perfringens (C.perfringens), formerly known as Clostridium welchii (C.welchii) or Bacillus perfringens (Bacillus perfringens), is a cause of necrotic enteritis, enterotoxemia in various animals, food poisoning in humans It is one of the main pathogens of traumatic gas gangrene and is widely distributed in the soil and the digestive tract of animals. The bacteria can produce highly toxic exotoxins and some enzymes related to invasiveness, and some strains can also produce enterotoxin, hemolysin and hemagglutinin. Traditionally, according to the difference of the four main exotoxins it produces, it can be divided into A (α), B (α, β, ε), C (α, β), D (α, ε), E (α , ι) 5 types (Lu Chengping. Veterinary Microbiology [M]. Beijing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/02C07K14/33A61K39/08A61K39/39A61P31/04C12R1/145
Inventor 彭小兵彭国瑞李旭妮蒋玉文
Owner CHINA INST OF VETERINARY DRUG CONTROL
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