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Clostridium septicum alpha toxin recombinant subunit vaccine and production method thereof

A subunit vaccine, Clostridium putrefaction technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, vaccines, etc., can solve the problems of local inflammation and toxicity, immune failure, immune effect decline, etc., to reduce biosafety risk effect

Active Publication Date: 2018-03-20
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The currently used commercial vaccines are mainly inactivated vaccines. Although they have achieved certain effects in preventing diseases caused by Clostridium putrefaciens, these vaccines still have some defects in the use process, for example, vaccine immunization is easy to cause local inflammation in animals and toxic reactions, etc.; the inactivation of exotoxins is involved in the preparation process, and there are biological safety hazards such as toxin leakage or incomplete inactivation; in addition, various microtoxins and bacterial metabolites in the culture supernatant often become immune Animal allergens, vaccinated animals are prone to adverse reactions, resulting in decreased immune effect or even immune failure

Method used

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  • Clostridium septicum alpha toxin recombinant subunit vaccine and production method thereof
  • Clostridium septicum alpha toxin recombinant subunit vaccine and production method thereof
  • Clostridium septicum alpha toxin recombinant subunit vaccine and production method thereof

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Experimental program
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Effect test

Embodiment 1

[0046] ——Construction, expression and identification of α-toxin 4 amino acid mutants of Clostridium putrefaciens

[0047] 1. Gene synthesis

[0048] According to the sequence of the natural protein gene of Clostridium putrefacilis α-toxin, this application designs 4 amino acid mutations after codon optimization, respectively mutating the 54th cysteine ​​of the mature toxin of the wild-type Clostridium putrefactive α-toxin into leucine acid, asparagine at position 264 is mutated to alanine, histidine at position 269 is mutated to alanine, and tryptophan at position 310 is mutated to alanine, thus obtaining Clostridium putrefaciens which is non-toxic to animals Alpha toxin mutants. At the same time, a 6-histidine tag was added to the C-terminus of the mutant protein. The gene sequence was synthesized by chemical synthesis method, which contains 1254 nucleotides in total. Among them, positions 1-1233 are the mature toxin sequence of Clostridium putrefaction alpha toxin (includ...

Embodiment 2

[0064] ——Toxicity test of α-toxin 4 amino acid mutant of Clostridium putrefaciens to mice

[0065] The toxicity of the α-toxin 4 amino acid mutant of Clostridium putrefaction to mice was determined to verify the actual attenuation effect of the mutant in vivo. The Clostridium putrefaction α-toxin 4 amino acid mutant recombinant protein mCSA activated by Clostridium putrefaction α-toxin 4 amino acid mutant, and the wild-type Clostridium putrefaction α-toxin were inoculated into 16-18g mice via the tail vein at different doses, each Inject 5 rats at a dose of 0.2ml / rat. Results When the inoculation dose was 0.1 mg, all the mice were alive and without adverse reactions, while the wild-type control group could cause 5 / 5 mice to die when inoculated with 10 ng. This result indicated that the C. putrefaciens alpha toxin mutant was avirulent in mice and was identified as a toxin avirulent mutant.

[0066] Table 1 Toxicity of recombinant protein mCSA to mice

[0067]

Embodiment 3

[0069]——Immunogenicity test of α-toxin 4 amino acid mutants of Clostridium putrefaciens

[0070] (1) Bacterial liquid culture: the Escherichia coli BL21 / mCSA strain culture liquid of the recombinant expression Clostridium putrefaction α toxin mutant protein is inoculated with LB liquid culture containing kanamycin by 2% of the total amount of the culture medium base, cultured in a fermenter. The culture parameters were set as follows: culture temperature 37°C, pH value 7.0, dissolved oxygen 40%. when culture OD 600 When the value is 10-15, lower the temperature to 28°C, and add IPTG with a final concentration of 0.3mmol / L to induce culture for 4h.

[0071] (2) Bacteria destruction: collect the bacteria by centrifugation, add 10ml of lysate (pH 7.2 0.02mol / LTris buffer, 0.3mol / L NaCl) to resuspend the bacteria according to the wet weight of each gram of the bacteria, and resuspend the bacteria under the conditions of 4 Use a low-temperature high-pressure homogenizer to disru...

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Abstract

The invention relates to a clostridium septicum alpha toxin recombinant subunit vaccine and a production method thereof. According to the production method, 54-site cysteine of wild clostridium septicum alpha mature toxin is mutated into leucine, 264-site asparaginate is mutated into alanine, 269-site histidine is mutated into alanine, and 310-site tryptophan is mutated into alanine, so that an alpha toxin mutant which is nontoxic to animals is obtained; and 6 groups of histidine tags are added to a C end of a mutant protein so as to obtain an antigen for preparing vaccines. The invention simultaneously discloses a recombinant expression vector and a recombinant host cell which contain an encoding gene of a nontoxic alpha toxin mutant of clostridium septicum. The efficacies of the preparedclostridium septicum alpha toxin recombinant subunit vaccine are far higher than the efficacies of existing vaccines, and the bio-safety risk in the production process of the vaccine is greatly reduced. Besides, by virtue of the superiority that a semi-finished product of the vaccine is high in protein concentration, a mixed vaccine can be prepared without increasing the dosage of the vaccine.

Description

technical field [0001] The invention relates to a Clostridium putrefaction alpha toxin recombinant subunit vaccine and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium septium is a Gram-positive anaerobic bacillus that can cause malignant edema, muscle necrosis, gas gangrene, and necrotic enteritis in animals and humans. Sheep are infected with the bacteria through the digestive tract. The sheep rapid disease (Braxy) caused by it is a common, multiple, non-contact, acute and fatal infectious disease. The bacteria can secrete a variety of exotoxins such as α, β, γ and δ, among which α toxin is its main pathogenic virulence factor and immune protective antigen. This toxin has three biological activities of hemolysis, lethality and necrosis, and can Inflammation and necrosis of the mucous membrane of the digestive tract (especially the mucous membrane of the abomasum), and at the same time enteri...

Claims

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Application Information

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IPC IPC(8): A61K39/08A61K39/39A61P31/04C12P21/02C12R1/19
CPCA61K39/08A61K39/39A61K2039/552A61K2039/575C07K14/33
Inventor 陈小云杜吉革薛麒朱真王磊印春生李启红康凯姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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