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Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine

A technology of Clostridium perfringens and mutants, which is applied in the direction of anti-bacterial immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc. It can solve the problems of poor immunogenicity and protein structure changes, etc. Achieve the effects of toxin safety, easy purification, and ease of complex production processes

Active Publication Date: 2022-05-31
天康制药股份有限公司
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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 a fusogenic tag was added at the same time, resulting in changes in the protein structure and poor immunogenicity; at the same time, the ε toxin recombinant protein was expressed in the cell and required ultrasonic disruption and nickel column purification and other complex production processes

Method used

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  • Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine
  • Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine
  • Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

In the PRB373 plasmid, the sequencing was correct, and the expression plasmid PYL was obtained. The structural map of the plasmid is shown in Figure 8.

2. obtain target gene:

Optimal design is carried out to the ε toxin encoding gene of Clostridium perfringens, and the 151st histidine is mutated to alanine

Acid, obtained gene fragment through artificial synthesis, the nucleotide sequence is SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.1

Show.

Upstream primer sequence is: 5'-AAACTATGAAAAAAAATCTTGTAAAA-3' (SEQ ID NO.4);

[0076] The downstream primer sequence is: 5'-TTGGCGCGCCTTATTTTATTCCTGGTGCCTT-3' (SEQ ID NO.5).

PCR amplification system is as follows:

[0078]

DNA Temple1.0μl

[0080]

Primer 20.5μl

Premix Ex Taq 12.5μl

ddH

Embodiment 2

Embodiment 3

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Abstract

The invention provides a Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine, and relates to the field of biotechnology. The Clostridium perfringens epsilon toxin mutant protein includes an amino acid sequence such as SEQ ID The 151st amino acid of Clostridium perfringens epsilon toxin shown in NO.3 was mutated to alanine. Compared with the wild type, the Clostridium perfringens epsilon toxin mutant protein toxicity is significantly reduced and has good immunogenicity. The vaccine can alleviate technical problems such as complex production process, high cost, complex antigen, batch-to-batch variation and large side effects of inactivated vaccines.

Description

Clostridium perfringens ε toxin mutant protein and preparation method, application and vaccine thereof technical field The present invention relates to the field of biotechnology, particularly to a kind of Clostridium perfringens epsilon toxin mutant protein and its Preparation methods, applications and vaccines. Background technique Clostridium perfringens (Clostridium Perfringens), also known as Clostridium welchii, is an important zoonotic Disease, Clostridium perfringens can cause traumatic gas gangrene and food poisoning in humans, and can also cause necrotizing Enteritis, sheep diarrhoea, cattle and sheep enterotoxemia and lamb dysentery are therefore a huge threat to animal husbandry. Clostridium perfringens main To secrete four exotoxins, α, β, ε, and ι, the types of exotoxins secreted are different. Clostridium perfringens can be divided into A, B, There are five toxin types C, D and E. Clostridium perfringens has the characteristics of acute onset, sho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/33C12N15/31C12N15/74C12N15/75C12N15/77C12N1/21C07K16/12G01N33/569A61P31/04A61K39/08C12R1/45
CPCC07K14/33C12N15/74C07K16/1282G01N33/56911C12N15/75C12N15/77A61K39/08A61P31/04G01N2333/33Y02A50/30
Inventor 贺笋王钢曹剑黎明宋江霞张瑾瑜高杨毅王鹏江袁科
Owner 天康制药股份有限公司
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