Clostridium perfringens epsilon toxin recombinant subunit vaccine and production method thereof

A Clostridium perfringens, subunit vaccine technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, vaccines, etc., can solve the large-scale production of vaccines biosafety risks, incomplete antigenic protein antigenicity, impact Immunogenicity and other issues, to avoid the impact of antigenic protein immunogenicity, reduce preparation time and production costs, and maintain the effect of immunogenicity

Active Publication Date: 2018-03-06
CHINA INST OF VETERINARY DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there have been reports of attenuated mutants of Clostridium perfringens epsilon toxin, such as Yu Li et al. ("Clostridium perfringens epsilon toxin attenuated mutants and their applications", application No.: 201410707351.6) mutated the tyrosine at position 71 of the mature toxin of the wild-type Clostridium perfringens epsilon toxin into a non-aromatic amino acid, and obtained the epsilon toxin mutant attenuated for cells or animals. Since this application only Mutation of 1 amino acid is prone to reverse mutation and becomes a highly virulent toxin again, which will bring serious

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0050] ——Construction, expression and identification of Escherichia coli BL21 / mETX-HYY strain (also known as Clostridium perfringens epsilon toxin 3 amino acid mutant in the present invention)

[0051] 1. Gene synthesis

[0052] According to the sequence of the natural protein gene of Clostridium perfringens epsilon toxin, the application designed three amino acid mutations after codon optimization, and the tyrosine at position 30 of the mature toxin of wild-type Clostridium perfringens epsilon toxin The acid is mutated to alanine, the 106th histidine is mutated to proline, and the 196th tyrosine is mutated to alanine, thereby obtaining the epsilon toxin mutant which is nontoxic to animals. The gene sequence of this section was synthesized by chemical synthesis method, which contains a total of 912 nucleotides. The specific nucleic acid sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0053] 2. Construction of non-fusion expression vec...

Embodiment 2

[0069] ——Toxicity test of Clostridium perfringens epsilon toxin avirulent mutant to mice

[0070] The toxicity of the avirulent mutant of Clostridium perfringens epsilon toxin to mice was tested to verify the actual attenuation effect of the mutant in vivo. The activated Clostridium perfringens ε toxin 3 amino acid mutant recombinant protein mETX-HYY, and the wild-type Clostridium perfringens ε toxin were inoculated into 16-18g mice via the tail vein at different doses, each dose Inject 5 rats, 0.2ml / rat. Results When the inoculation dose was 0.1mg, 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 1ng. This result indicated that the Clostridium perfringens epsilon toxin avirulent mutant was avirulent in mice and was identified as the toxin avirulent mutant.

[0071] Table 1 Toxicity of recombinant protein mETX-HYY to mice

[0072]

Embodiment 3

[0074] ——Immunogenicity test of Clostridium perfringens epsilon toxin 3 amino acid mutant

[0075] (1) Bacterial culture: the Escherichia coli genetically engineered bacterium BL21 / mETX-HYY strain culture liquid that recombinantly expresses Clostridium perfringens epsilon toxin protein is inoculated with kanamycin by 2% of the total amount of the culture medium. LB liquid medium, cultivated in 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 28°C, and add IPTG with a final concentration of 0.2mmol / L to induce culture for 10 hours.

[0076] (2) Bacteria destruction: 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 bacteria body, and resuspend the bacteria with high pressure The homogenizer crushed th...

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Abstract

The invention relates to a clostridium perfringens epsilon toxin recombinant subunit vaccine and a production method thereof. The clostridium perfringens epsilon toxin recombinant subunit vaccine prepared by the invention is produced by adopting recombinant clostridium perfringens epsilon toxin protein subjected to codon optimization and containing three amino acid mutations, and the integrality and a spatial conformation of the natural toxin protein are retained maximally, so that the immunogenicity is maintained, and the bio-safety risk brought by a single amino acid mutation is avoided. Thevaccine also has the advantages of simple preparation process, low immunizing dose, higher vaccine efficacy compared with an existing vaccine; compared with a currently commercial clostridium perfringens natural toxin inactivated vaccine in China, the bio-safety risk during a vaccine production process is greatly reduced, and the clostridium perfringens epsilon toxin recombinant subunit vaccine is an ideal candidate vaccine upgraded and updated based on a current D type clostridium perfringens toxin vaccine in China; and when the vaccine and other antigens are used for jointly preparing a mixed vaccine, the mixed vaccine can be prepared without the need for increasing the dosage of the mixed vaccine.

Description

technical field [0001] The invention relates to a Clostridium perfringens epsilon 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 major lethal exotoxins α, β, ε and ι produced, the bacteria were divided into five toxin types A, B, C, D and E. Clostridium perfringens has the characteristics of...

Claims

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Application Information

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IPC IPC(8): A61K39/08A61K39/39A61P31/04C12N1/21C12R1/19
CPCA61K39/08A61K39/39A61K2039/552A61K2039/575C07K14/33
Inventor 陈小云杜吉革朱真薛麒王磊印春生李启红康凯姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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