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Clostridium perfringens Beta toxin recombination subunit vaccine and production method thereof

A technology for Clostridium perfringens and subunit vaccines, which can be applied to microorganism-based methods, biochemical equipment and methods, vaccines, etc. Conformation, the effect of avoiding the effects of antigenic protein immunogenicity

Active Publication Date: 2018-12-25
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 recombination subunit vaccine and production method thereof
  • Clostridium perfringens Beta toxin recombination subunit vaccine and production method thereof
  • Clostridium perfringens Beta toxin recombination subunit vaccine and production method thereof

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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 beta toxin, the present invention designs 4 amino acid mutations after codon optimization, respectively, to replace the 212th spermine of the wild-type Clostridium perfringens beta toxin mature toxin. The acid was mutated to glutamic acid, the leucine at position 268 was mutated to glycine, and the tyrosine at position 266 and tryptophan at position 278 were mutated to alanine, thereby obtaining a beta toxin mutant that is non-toxic to animals. . At the same time, add MBP tags and 6×His tags to the N-end and C-end respectively. Using chemical synthesis, the gene sequence GMBPCPB was synthesized m4 , Contains a total of 2097 nucleotides.

[0067] The specific nucleic acid sequence is shown...

Embodiment 2

[0092] ——The virulence test of the non-toxic mutant of Clostridium perfringens beta toxin on mice

[0093] The virulence of the non-toxic mutant of Clostridium perfringens beta toxin to mice was tested to verify the actual attenuation effect of the mutant in vivo. The purified C. perfringens beta toxin 4 amino acid mutant recombinant protein mMBPCPB m4 , And C-type Clostridium perfringens culture supernatant, respectively, with different doses, 16-18g mice were inoculated through the tail vein, 5 mice per dose, 0.2mL / mouse. Results When the inoculation dose was 0.1 mg, all mice were alive and had no adverse reactions, while the wild-type control group could cause 5 / 5 death of mice when inoculated with 0.001 mL. This result indicates that the non-toxic mutant of Clostridium perfringens β-toxin is non-toxic to mice and was determined to be a non-toxic mutant of the toxin.

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

[0095]

Embodiment 3

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

[0098] 1. Bacteria culture

[0099] The culture broth of Escherichia coli genetically engineered bacterium BLC2 strain recombinantly expressing the β-toxin protein of Clostridium perfringens was inoculated with LB liquid medium containing kanamycin at 2% of the total medium, and cultured in a fermenter. Set the culture parameters as follows: culture temperature 37°C, pH value 7.0, and dissolved oxygen higher than 20%. When the culture OD 600 When the value is 10-15, the temperature is lowered to 15°C, and IPTG with a final concentration of 0.5 mmol / L is added to induce culture for 16 hours.

[0100] 2. Broken bacteria

[0101] Collect the bacteria by centrifugation, add 10mL lysate (0.02mol / LTris buffer (pH 7.2), 0.3mol / LNaCl) per gram of bacteria to resuspend the bacteria, and crush them with a high-pressure homogenizer at a pressure of 800bar The cells were centrifuged to collect the super...

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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 recombinant subunit vaccine of Clostridium perfringens beta toxin and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium perfringens, also known as Clostridium wilfordii, is an important zoonotic disease. The pathogen is traumatic gas gangrene and human food poisoning, as well as fast epidemic disease of sheep, lamb dysentery, cattle and sheep necrotizing enteritis, cattle One of the main pathogens of enterotoxemia in sheep has caused huge economic losses to the livestock industry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. The bacteria can produce at least 18 exotoxins and exert pathogenic effects through the toxins it produces. According to the types of the four main lethal exotoxins α, β, γ and ι, the bacteria were divided into five toxin types A, B, C, D, and E. Clostridium perfringens disease has the ch...

Claims

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Application Information

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Patent Type & Authority Applications(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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