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Veterinary D-type clostridium perfringen toxin, and preparation method and special culture medium of D-type clostridium perfringen toxin

A technology of Clostridium perfringens and toxin-producing medium, which is applied in biochemical equipment and methods, veterinary vaccines, chemical instruments and methods, etc., and can solve the problems of unstable toxin-producing performance, waste of vaccine production, and high cost To achieve the effects of stable toxin-producing performance, convenient preparation and use, and strong toxin-producing ability

Active Publication Date: 2017-10-27
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the vaccines currently on the market generally use the enzymatic digestion solution of beef and liver as raw materials to prepare the medium. The preparation process of the medium prepared by this method is cumbersome, time-consuming, and requires a lot of manpower. Uneven quality leads to unstable toxin production performance, which affects the quality of the vaccine, and also brings great waste and high cost to vaccine production

Method used

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  • Veterinary D-type clostridium perfringen toxin, and preparation method and special culture medium of D-type clostridium perfringen toxin
  • Veterinary D-type clostridium perfringen toxin, and preparation method and special culture medium of D-type clostridium perfringen toxin
  • Veterinary D-type clostridium perfringen toxin, and preparation method and special culture medium of D-type clostridium perfringen toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the screening of Type D Clostridium perfringens toxin-producing medium

[0048] (1) Three different medium formulations were designed:

[0049] Formula 1: Soybean peptone 15g, casein peptone 15g, yeast extract powder 5g, glucose 5g, purified water to 1000mL.

[0050] Formula 2: Show peptone 10g, casein peptone 10g, yeast extract powder 15g, sodium chloride 4g, sodium carbonate 0.6g, calcium chloride 0.1g, cystine 2g, glucose 10g, add purified water to 1000mL.

[0051] Formula 3: Soybean peptone 10g, casein peptone 10g, yeast extract powder 5g, Na 2 HPO 4 12H 2 O 5g, dextrin 10g, purified water added to 1000mL.

[0052] (2) Prepare culture medium

[0053] Except for dextrin and glucose, weigh or measure each component according to the above content, add purified water, heat to fully dissolve, add purified water to make it to the final volume required for preparation, adjust with 10M sodium hydroxide pH value to 7.5 ~ 8.0. Add dextrin to formula 3 acc...

Embodiment 2

[0066] Embodiment 2, the optimization of the preparation of Clostridium perfringens type D toxin-producing medium and the method of use

[0067] (1) Optimization of Erlenmeyer flask culture conditions

[0068] The culture temperature, initial pH value, and culture time were optimized by means of static culture in the triangular flask, and the optimal conditions were determined to be "the initial pH value of the medium was 8.0-8.5, and cultured at 35-37°C for 17-18 hours".

[0069] The virulence result table of Erlenmeyer flask culture with optimized conditions

[0070]

[0071]

[0072] As can be seen from the above table, according to the method of the present invention, the highest virulence can be raised to 10 times of the seedling production standard of the regulations.

[0073] (2) Optimization of fermentation tank culture conditions

[0074] The control of pH value, culture time, and sugar supplementation were optimized by means of fermentation tank culture, and ...

Embodiment 3

[0078] Example 3, Preparation and Efficacy Evaluation of Type D Clostridium perfringens Toxoid Vaccine

[0079] Take the cultured bacterial culture, add 0.7% formaldehyde solution (40%) by volume, and put it at 35°C for inactivation and detoxification for 5 days. Take the inactivated and detoxified bacteria solution to inoculate anaerobic broth, common broth, and common agar slant, and observe the sterile growth for 5 days, indicating that the inactivation is complete; at the same time, the inactivated and detoxified bacteria solution is centrifuged at 3000r / min for 30min, and the bacteria are discarded Keep the supernatant from the precipitate, filter the supernatant with a 0.22 μm filter membrane, inject 2 mice with a weight of 16-18 g into the tail vein, and inject 0.4 ml into each mouse, and observe the health and life for 3 days, indicating that the detoxification is complete.

[0080] Take the inactivated and detoxified bacterial solution, centrifuge at 3000r / min for 30m...

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Abstract

The invention discloses a preparation method and a special culture medium of veterinary D-type clostridium perfringen toxin. The culture medium per 100ml comprises the following components: 1-1.5g soy peptone, 1-1.5g casein peptone, 0.5-0.75g yeast extract, 0.5-0.75g Na2HPO4.12H2O, 1-1.5g dextrin and the balance of water, wherein a pH (potential of hydrogen) value of the culture medium is 8.0-8.5. The preparation method of the D-type clostridium perfringen toxin comprises the steps of inoculating a D-type clostridium perfringen production strain into the culture medium, collecting a culture material, performing centrifugation, and then filtering a supernate. With the adoption of the method, the maximum toxicity can be improved to be 45 times a seedling standard in Regulations on National Veterinary Biological Products, and an output-input ratio can be increased to be 30-225 times that of the original traditional technology. In addition, corresponding serum neutralization titer of a toxoid vaccine prepared by the D-type clostridium perfringen toxin on a rabbit and a sheep is also improved to be 8.3 and 13.3 times a regulation standard respectively.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a veterinary D-type Clostridium perfringens toxin, a preparation method thereof and a special culture medium. Background technique [0002] Sheep rapid disease, sudden attack, lamb dysentery and enterotoxemia are caused by Clostridium putrefaciens, Clostridium perfringens type C, Clostridium perfringens type B and Clostridium perfringens type D respectively. Common multiple infectious diseases [1, Lu Chengping. Veterinary Microbiology [M]. Beijing: China Agricultural Press, 2013: 192-202.], they often occur in combination, the course of the disease is sharp, and the affected animals often die without showing symptoms. The mortality rate is high and the harm is great. Therefore, immunization is the only effective way to control these diseases. Developed animal husbandry such as Europe, America, Australia, etc. all regard the four diseases of cattle and s...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/02C07K14/33A61K39/08A61P31/04C12R1/145
CPCA61K39/08A61K2039/552A61K2039/70C07K14/33C12N1/20C12P21/02
Inventor 蒋玉文彭小兵李旭妮彭国瑞
Owner CHINA INST OF VETERINARY DRUG CONTROL
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