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Clostridium septicum alpha toxin genetic engineering vaccine and production method thereof

A genetically engineered vaccine, the technology of Clostridium putrefaciens, which is applied in the field of Clostridium putrefactive alpha toxin genetically engineered vaccines, can solve the problems of loss of cytotoxicity and unverified antigenicity of recombinant toxins, etc., and achieve the effect of reducing biosafety risks

Active Publication Date: 2019-03-01
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have found that deletion of 11 amino acids (212-222) in the transmembrane region of the amphipathic β hairpin located in the D2 region does not significantly change the normal folding of CSA, but completely loses the cytotoxicity and Its role in Clostridium putrefaction, while the antigenicity of this recombinant toxin has not been verified

Method used

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  • Clostridium septicum alpha toxin genetic engineering vaccine and production method thereof
  • Clostridium septicum alpha toxin genetic engineering vaccine and production method thereof
  • Clostridium septicum alpha toxin genetic engineering vaccine and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] ——CSA mutants containing both 4 amino acid mutations and 11 amino acid deletions (rCSA M4Δ11 ) construction, expression and identification of expression vector

[0047] 1. Gene synthesis

[0048] According to the gene sequence of natural CSA, the applicant designed 4 amino acid mutations (C54L, N264A, H269A and W310A) and 11 amino acid deletions (positions 212-222) after codon optimization, thereby obtaining the avirulent rCSA M4Δ11 . At the same time, 6 histidine tags were added to the C-terminus of the recombinant protein. The gene sequence was synthesized by chemical synthesis method, which contains 1221 nucleotides in total. Among them, positions 1-1200 are CSA mature toxin sequences (including 4 amino acid mutations and 11 amino acid deletions), and positions 1201-1218 are 6 histidine tag sequences. The specific nucleic acid sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0049]

[0050]

[0051]

[0052] 2. C...

Embodiment 2

[0067] ——rCSA M4Δ11 Toxicity test on mice

[0068] rCSA M4Δ11 Toxicity to mice to verify the actual attenuation effect of the mutant in animals. rCSA M4Δ11 As well as recombinant CSA (rCSA) without amino acid mutation, 16-18g mice were inoculated through the tail vein at different doses, 5 mice per dose, 0.2mL / mouse. Results When the inoculation dose was 0.1mg, all the mice were healthy and had no adverse reaction, while rCSA inoculation of 46.88ng could cause the death of 5 / 5 mice. The results indicated that rCSA M4Δ11 It is avirulent in mice and was identified as an avirulent mutant of CSA.

[0069] Table 1 rCSA M4Δ11 Toxicity to mice

[0070]

Embodiment 3

[0072] ——rCSA M4Δ11 immunogenicity test

[0073] (1) Bacteria culture: the recombinant expression rCSA M4Δ11 Escherichia coli BLc1 strain culture liquid, inoculated with LB liquid medium containing kanamycin according to 2% of the total amount of the culture medium, and 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, add IPTG with a final concentration of 0.5mmol / L to induce culture for 4h.

[0074] (2) Bacterial destruction: collect the bacteria by centrifugation, add 10mL of lysate (pH value 7.2, 0.02mol / L Tris buffer, 0.3mol / L NaCl) to resuspend the bacteria according to the wet weight of each gram of the bacteria, and resuspend the bacteria at 4°C. Under the condition of low temperature and high pressure homogenizer with the pressure of 800bar, the bacterium was broken 3 times. The lysate was centrifuged at 10,000 r / min at 4°C for 30 min, the...

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Abstract

The invention provides a prepared clostridium septicum CSA genetic engineering vaccine, which is optimized by a codon and is produced by recombinant clostridium septicum CSA (rCSAM4 delta 11) obtainedby deleting 11 amino acids (212th bit to 222nd bit) on the basis of containing 4 amino acid mutations (C54L, N264A, H269A and W310A); the completeness and spatial conformation of the natural toxin are reserved to the maximum degree, so that the immunogenicity is maintained; the biological safety hazards due to minority amino acid mutation are avoided. Meanwhile, nontoxic rCSAM4 delta 11 can be expressed in a soluble form, so that the influence of the complicated process of the inclusion body denaturation and renaturation on antigen protein immunogenicity is avoided, and the preparation time and the production cost of the vaccine are reduced. In addition, the vaccine also has the advantages of low immunizing dose, good immunizing effect and the like, and belongs to an ideal candidate vaccine for upgrading and updating the existing clostridium septicum toxin vaccine.

Description

technical field [0001] The invention relates to a Clostridium putrefactive alpha toxin genetic engineering vaccine and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium putrefaciens is an anaerobic bacterium that can cause disease in humans, cattle, sheep, horses, pigs, minks, chickens, etc. It is extremely harmful to human health and livestock and poultry breeding. Bacteria caused by Braxy is a common, multiple, non-contact, acute and fatal infectious disease. Due to the short duration of the disease caused by Clostridium putrefaction, if the symptoms are not treated in time, the animals will die quickly, and immunization is the most effective way to prevent this kind of disease. At present, the commercialized vaccines used are mainly inactivated vaccines. Although they have achieved certain effects in preventing diseases caused by Clostridium putrefaciens, these vaccines still have some defec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/08A61P31/04
CPCA61K39/08A61K2039/552A61P31/04
Inventor 杜吉革陈小云薛麒朱真李启红印春生康凯姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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