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Clostridium perfringens beta toxin mutant protein as well as preparation method, application and vaccine thereof

A technology of Clostridium perfringens and β-toxin, which is applied in the direction of anti-bacterial immunoglobulin, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of protein structure changes and poor immunogenicity, etc. Achieve the effect of good effectiveness, simple process and avoid complex production process

Active Publication Date: 2020-11-17
天康制药股份有限公司
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  • 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 a fusion-promoting tag was added at the same time, resulting in changes in the protein structure and poor immunogenicity; at the same time, the β-toxin recombinant protein was expressed in the cell and required ultrasonic disruption and nickel column purification and other complex processes

Method used

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  • Clostridium perfringens beta toxin mutant protein as well as preparation method, application and vaccine thereof
  • Clostridium perfringens beta toxin mutant protein as well as preparation method, application and vaccine thereof
  • Clostridium perfringens beta toxin mutant protein as well as preparation method, application and vaccine thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1. Construction of pYL expression plasmid:

[0061] 1.1 Synthetic promoter, ori region, and Erm resistance gene: According to the requirements for expression vectors, Xyl / tet fragments, ori regions, and Erm resistance genes that can be screened with antibiotics are artificially chemically synthesized using tetracycline for expression regulation.

[0062] 1.2 Construction of PYL expression plasmid: Insert the gene fragment synthesized in the above 1.1 into the PRB373 plasmid by molecular biology method, the sequence is correct, and the expression plasmid PYL is obtained. The structural map of the plasmid is as follows Figure 8 shown.

[0063] 2. Obtain the target gene:

[0064] The wild-type Clostridium perfringens beta toxin coding gene is optimally designed, and the nucleotide sequence of the wild-type Clostridium perfringens beta toxin is shown in SEQ ID NO.6 (containing signal peptide), which is mutated , the obtained nucleotide sequence is shown in SEQID NO.5 (cont...

Embodiment 2

[0079] Expression and identification of Clostridium perfringens beta toxin mutant protein: the recombinant strain SE / PYL-CPB prepared in the examples R239A-Y293A Inoculate in TSB liquid medium (containing 5 μg / ml Erm), the inoculation ratio is 1%, and the inducer ATC with a final concentration of 500ng / ml is used, and the culture is shaken at 200r / min under the condition of temperature 36~37℃ for 18~ 24 hours.

[0080] The prepared recombinant strain was inoculated into TSB liquid medium containing 5 μg / ml Erm at a ratio of 1%, and the inducer ATC was added at 500 ng / ml, placed in a constant temperature shaking incubator at 36-37°C, and cultured at 200 r / min for 18 hours. After ~24 hours, the culture was centrifuged at high speed to collect the supernatant, which was identified by SDS-PAGE electrophoresis and Western Blot.

[0081] SDS-PAGE identification: take the supernatant for SDS-PAGE electrophoresis, observe and record the protein content and protein purity of the Clost...

Embodiment 3

[0084] The mouse toxicity test of Clostridium perfringens beta toxin mutant protein, the experimental method refers to the three regulations of "The Veterinary Pharmacopoeia of the People's Republic of China" (2015 edition). The experimental animals were 18±2g mice, 5 in each group, and randomly divided into six groups. The experimental groups are as follows: three experimental groups, immunized mice with three injection doses of 1 μg, 10 μg and 100 μg respectively; the negative control is the culture medium; the positive control is a natural toxin of MLD; and the blank control. The sample was diluted with gelatin buffer, and the sample was injected into the tail vein with a total volume of 200 μL. The mice were observed for 1 to 3 days and recorded for death. Experimental results: All the mice in the negative control group and the experimental group survived, and all the mice in the positive control group died.

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Abstract

The invention provides clostridium perfringens beta toxin mutant protein as well as a preparation method, application and a vaccine thereof, and relates to the field of biological products. The clostridium perfringens beta toxin mutant protein is prepared by mutating 212th arginine and 266th tyrosine of wild clostridium perfringens beta toxin with an amino acid sequence as shown in SEQ ID NO.3 into alanine, and the toxicity of the clostridium perfringens beta toxin mutant protein is remarkably reduced compared with that of the wild clostridium perfringens beta toxin. Meanwhile, good immunogenicity is also reserved.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a Clostridium perfringens beta toxin mutant protein and its preparation method, application and vaccine. Background technique [0002] Clostridium perfringens (Clostridium Perfringens), also known as Clostridium perfringens or Clostridium welchii, is the most common Clostridium among gas gangrene pathogens clinically, it can decompose sugar in muscle and connective tissue, produce A large amount of gas causes severe emphysema of the tissue, which in turn affects the blood supply and causes extensive necrosis of the tissue. Clostridium perfringens is also one of the main pathogens of sheep blight, lamb dysentery, cattle and sheep necrotic enteritis, and cattle and sheep enterotoxemia, which has caused huge economic losses to animal husbandry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. According to the types of four major lethal exotox...

Claims

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Application Information

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IPC IPC(8): C07K14/33C07K16/12C12N15/31C12N15/74C12N15/75C12N15/77G01N33/569A61K39/08A61P31/04
CPCC07K14/33C07K16/1282C12N15/74C12N15/75C12N15/77G01N33/56911A61K39/08A61P31/04G01N2333/33Y02A50/30
Inventor 贺笋王钢候凤张慧敏周涛刘强德鲍子磊张艳盟姜峰
Owner 天康制药股份有限公司
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