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Clostridium perfringen alpha toxin genetic engineering vaccine and preparation method thereof

A technology for Clostridium perfringens, genetically engineered vaccines, applied in the directions of genetic engineering, microorganism-based methods, and botanical equipment and methods

Active Publication Date: 2019-04-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In these reports, IPTG was used as an inducer to express in the E. coli system, and it was necessary to detect the growth of the recombinant bacteria and add an inducer; the α-toxin recombinant protein was expressed intracellularly, requiring complex processes such as ultrasonication and nickel column purification.

Method used

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  • Clostridium perfringen alpha toxin genetic engineering vaccine and preparation method thereof
  • Clostridium perfringen alpha toxin genetic engineering vaccine and preparation method thereof
  • Clostridium perfringen alpha toxin genetic engineering vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Construction of Escherichia coli BL21(DE3) / pET32a-αH176N strain

[0087] 1. Determination of α-toxin gene sequence

[0088] (1) According to the α-toxin gene sequence published on GeneBank (GenBank: AY823400.1), primers were designed as follows:

[0089] Upstream primer: 5-GCGGAATTCATGAAAAGAAAGATTTGTAAG-3 (EcoR I)

[0090] Downstream primer: 5-GCGCTCGAGTTATTTTTATATTATAAGTTG-3(Xho I)

[0091] (2) Using Clostridium perfringens type A (Su 84-A strain) DNA as a template, PCR reaction system: 1 μL template, 0.2 μL high-fidelity Taq enzyme, 5 μL 10×Taq Buffer, upstream and downstream primers (20 μmol / L) each 0.5μL, dNTPs (25mmol / L) 4μL, MgSO 4 (50mmol / L) 2μL, ddH2 O 37 μL.

[0092] 94°C for 2min→(94°C for 30s→62.0°C for 1min→68°C for 1.5min) for 30 cycles→68°C for 10min.

[0093] (3) After the PCR amplified product was subjected to agarose gel electrophoresis, the target fragment was recovered with a gel recovery kit. The recovered target fragments were sent ...

Embodiment 2

[0101] The optimization of embodiment 2 self-inducing conditions

[0102] 1. Preparation of autoinduction medium containing different doses of lactose

[0103] Medium preparation: 10g / L tryptone, 5g / L yeast extract, 5g / L glycerol, 0.25g / L glucose, 25mM Na 2 HPO 4 , 25mM KH 2 PO 4 , 50mM NH 4 CL, 5mM Na 2 SO 4 , 2mM Mg 2 SO 4 , and 1g / L, 2g / L, 4g / L lactose, sterilized at 121°C for 15min, to prepare autoinduction medium containing 1g / L, 2g / L, 4g / L lactose.

[0104] 2. Optimization of self-induction conditions

[0105] Escherichia coli BL21(DE3) / pET32a-αH176N strain was inoculated at 1% in the autoinduction medium containing 1g / L, 2g / L, 4g / L lactose, and the culture medium of each lactose concentration was at 16°C and 28°C respectively. , After 24 hours of shaking culture at 37°C, centrifuge at 6000r / min for 15 minutes, collect the supernatant, filter through a 0.22μm pore size filter, add 4×SDS loading buffer to mix, boil for 5-8 minutes, and carry out 12% SDS- PAGE a...

Embodiment 3

[0106] Example 3 Toxicity determination of recombinant α-toxin αH176N protein

[0107] 1. Lecithin enzyme activity test

[0108] α-toxin has phospholipase C activity, which can specifically hydrolyze phospholecithin in egg yolk into phosphorylcholine and 1,2-diglyceride, resulting in white turbidity. Add 2 μL of the αH176N protein and the wild-type α-toxin in Example 1 to the egg yolk agar plate respectively. No white turbid spots appear in the position, indicating that the αH176N protein no longer possesses the phospholipase C activity of α toxin.

[0109] 2. Toxicity test in mice

[0110] Ten 16-20g mice were randomly divided into 2 groups, 5 in each group. One of them was used as a control group, and the whole bacterial solution of the empty vector Escherichia coli BL21(DE3) / pET32a self-induction was centrifuged and the supernatant was taken, and the filtrate of the 0.22 μm pore-diameter filter was injected into the tail vein of 0.3ml / mouse; Another group of mice was in...

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Abstract

The invention provides a clostridium perfringen alpha toxin genetic engineering vaccine and a preparation method thereof. Compared with wild type alpha toxins, clostridium perfringen alpha toxin recombination protein is characterized in that the 176th site histidine of an amino acid sequence is mutated to obtain asparagine. The clostridium perfringen alpha toxin genetic engineering vaccine is prepared from detoxifcation clostridium perfringen alpha toxin recombination protein of self-induction secretory expression. The vaccine has the advantages of being safer, better in immune efficacy, simpler in technology, lower in cost and the like, and can effectively solve the problems that in the prior art, the production technology of the vaccine is complexer, and the cost is higher.

Description

technical field [0001] The invention relates to a Clostridium perfringens alpha toxin genetic engineering vaccine and a preparation method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium perfringens (Clostridium Perfringens), also known as Clostridium welchii, is one of the main pathogenic bacteria that cause various animal necrotic enteritis, enterotoxemia, human and animal traumatic gas gangrene and human food poisoning. The pathogenic factor of the bacteria is the exotoxin secreted by it, mainly α, β, ε and ι toxin, and accordingly the bacterium is divided into 5 toxin types such as A, B, C, D, E, etc., and the ε toxin is B Type and D types of Clostridium perfringens are produced. Clostridium perfringens can cause fatal intestinal diseases in goats, sheep and other animals, causing great economic losses to animal husbandry. The onset of Clostridium perfringens is acute, the course of the disease is short, an...

Claims

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Application Information

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IPC IPC(8): A61K39/08A61K39/39A61P31/04C12N15/31C12N15/70C12N1/21C12R1/19
CPCA61K39/08A61K39/39A61P31/04C12N15/70C07K14/33A61K2039/55505
Inventor 魏后军王芳范志宇胡波宋艳华薛家宾仇汝龙
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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