Clostridium perfringens beta toxin recombinant subunit vaccine and production method thereof

A technology of Clostridium perfringens and subunit vaccines, applied in the direction of microorganism-based methods, biochemical equipment and methods, vaccines, etc., can solve the problems such as difficulty in increasing the renaturation rate, and achieve immunogenicity and high-efficiency expression , the effect of avoiding biological safety hazards

Active Publication Date: 2020-08-04
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and it is often difficult to improve the renaturation rate, which is the main constraint factor limiting its application.

Method used

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  • Clostridium perfringens beta toxin recombinant subunit vaccine and production method thereof
  • Clostridium perfringens beta toxin recombinant subunit vaccine and production method thereof
  • Clostridium perfringens beta toxin recombinant subunit vaccine and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] ——Construction, expression and identification of Escherichia coli BLC2 strain (also known as Clostridium perfringens beta toxin 4 amino acid mutant in the present invention)

[0065] 1. Gene synthesis

[0066] According to the sequence of the natural protein gene of Clostridium perfringens β-toxin, the present invention designs 4 amino acid mutations after codon optimization, and the 212th arginine of the mature toxin of wild-type Clostridium perfringens β-toxin Acid is mutated to glutamic acid, leucine at position 268 is mutated to glycine, tyrosine at position 266 and tryptophan at position 275 are mutated to alanine, thus obtaining a beta toxin mutant that is non-toxic to animals . At the same time, MBP tag and 6×His tag were added to N-terminal and C-terminal respectively. The gene sequence GMBPCPB was synthesized by chemical synthesis m4 , containing a total of 2097 nucleotides.

[0067] The specific nucleic acid sequence is shown in SEQ ID No.1,

[0068]

...

Embodiment 2

[0092] ——Toxicity test of Clostridium perfringens beta toxin avirulent mutant to mice

[0093] To verify the actual attenuation effect of the mutant in vivo by measuring the toxicity of the Clostridium perfringens beta toxin avirulent mutant to mice. Purified Clostridium perfringens beta toxin 4 amino acid mutant recombinant protein mMBPCPB m4 , and the culture supernatant of Clostridium perfringens type C, inoculate 16-18g mice via tail vein at different doses, 5 mice per dose, 0.2mL / mouse. Results When the inoculation dose was 0.1 mg, all the mice were healthy and had no adverse reactions, while the wild-type control group could cause 5 / 5 mice to die when inoculated with 0.001 mL. This result indicated that the Clostridium perfringens beta toxin avirulent mutant was avirulent to mice and was identified as the toxin avirulent mutant.

[0094] Table 1 Recombinant protein mMBPCPB m4 Toxicity to mice

[0095]

Embodiment 3

[0097] ——Immunogenicity test of Clostridium perfringens beta toxin 4 amino acid mutant

[0098] 1. Bacterial culture

[0099] Inoculate the LB liquid medium containing kanamycin according to 2% of the total amount of the culture medium of the Escherichia coli genetic engineering strain BLC2 strain recombinantly expressing Clostridium perfringens beta toxin protein, and cultivate in a fermenter. The culture parameters were set as follows: culture temperature 37°C, pH value 7.0, dissolved oxygen higher than 20%. when culture OD 600 When the value is 10-15, lower the temperature to 15°C, and add IPTG with a final concentration of 0.5mmol / L to induce culture for 16 hours.

[0100] 2. Broken bacteria

[0101] Collect the bacteria by centrifugation, add 10mL of lysate (0.02mol / L Tris buffer (pH value 7.2), 0.3mol / L NaCl) to resuspend the bacteria according to the weight of each gram of the bacteria, and use a high-pressure homogenizer at 800bar The cells were crushed under press...

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Abstract

The invention relates to a clostridium perfringens Beta toxin recombination subunit vaccine and a production method thereof. The prepared clostridium perfringens Beta toxin recombination subunit vaccine is produced by using a recombination clostridium perfringens Beta toxin protein which is processed by codon optimization and contains 4 amino acid mutations, namely integrity and spatial conformation of a natural toxin protein are reserved in the greatest degree, so immunogenicity of the natural toxin protein is kept, and a biological potential safety hazard caused by the single amino acid mutation is avoided. The vaccine further has the advantages of simple preparation process, low immunizing dose, good vaccine efficacy and the like. Compared with a current commercial clostridium perfringens natural toxin inactivated vaccine in China, a bio-safety risk in a vaccine production process is greatly reduced. The vaccine is a perfect candidate vaccine for updating a current C-type clostridium perfringens toxin vaccine in China. In addition, while a mixed vaccine is prepared by the vaccine with other antigens, the mixed vaccine can be prepared without increasing a using dose of the mixedvaccine.

Description

technical field [0001] The invention relates to a Clostridium perfringens beta toxin recombinant subunit vaccine and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium perfringens, also known as Clostridium welchii, is an important zoonotic disease, the pathogen is traumatic gas gangrene and human food poisoning and sheep plague, lamb dysentery, cattle and sheep necrotic enteritis, cattle It is one of the main pathogens of sheep enterotoxemia, which has caused huge economic losses to animal husbandry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. This bacterium can produce at least 18 kinds of exotoxins and play a pathogenic role through the toxins it produces. According to the types of four main lethal exotoxins α, β, γ and ι produced, the bacteria were divided into five toxin types A, B, C, D and E. Clostridium perfringens has the characteristics of acu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/08A61K39/39A61P31/04C12P21/02C07K19/00C07K1/30C12R1/19
CPCA61K39/08A61K39/39A61K2039/552A61K2039/55566A61P31/04C07K14/33C07K2319/21C07K2319/24
Inventor 杜吉革陈小云朱真薛麒刘莹印春生李启红康凯姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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