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

A technology for Clostridium perfringens and mutants, which is applied in antibacterial immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as protein structure changes and poor immunogenicity, etc. Achieve the effect of high expression amount and purity, simple production process, and small side effects of vaccines

Active Publication Date: 2020-11-20
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 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 as well as preparation method, application and vaccine thereof
  • Clostridium perfringens epsilon toxin mutant protein as well as preparation method, application and vaccine thereof
  • Clostridium perfringens epsilon toxin mutant protein as well as preparation method, application and vaccine thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] 1. Construction of pYL expression plasmid:

[0071] 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.

[0072] 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.

[0073] 2. Obtain the target gene:

[0074] The epsilon toxin coding gene of Clostridium perfringens was optimized and designed, and the 151st histidine was mutated into alanine, and the gene fragment was obtained through artificial synthesis. The nucleotide sequence is SEQ ID NO.2, and the amino acid sequence is as follows: Shown in SEQ...

Embodiment 2

[0085] Expression and identification of recombinant protein Clostridium perfringens epsilon toxin mutant protein:

[0086] 2.1 Prepare the recombinant strain SE / PYL-ETX H151A Inoculate TSB liquid medium containing 5 μg / ml Erm at a ratio of 0.5%, add inducer ATC at 400ng / ml, place in a constant temperature shaking incubator at 36-38°C, and shake at 200r / min for 18-36 hours.

[0087] 2.2 Collect the supernatant: collect the supernatant by high-speed centrifugation of the culture medium, perform SDS-PAGE detection, observe and record the protein content and protein purity of the recombinant protein Clostridium perfringens epsilon toxin mutant. see details image 3 , image 3 Middle lane M is the protein molecular weight marker; lanes a to d are 1000 μg / ml BSA, 500 μg / ml BSA, 250 μg / ml BSA and 125 μg / ml BSA in sequence, and lane 1 is recombinant Clostridium perfringens epsilon toxin mutant protein.

[0088] 2.3 Western Blot identification of recombinant protein: transfer the re...

Embodiment 3

[0090] Mouse toxicity test of recombinant protein Clostridium perfringens epsilon toxin mutant protein:

[0091] In this study, according to the regulations in the third part of "The Veterinary Pharmacopoeia of the People's Republic of China" (2015 edition), the method of tail vein injection was selected to detect the toxicity of Clostridium perfringens epsilon toxin mutants to mice. Mice weighing 16-20 g were randomly divided into 6 groups, 5 mice in each group, and injected with three injection doses of 1 μg, 10 μg and 100 μg respectively, and the culture medium was set as a negative control and a natural toxin of MLD was set as a positive control. The sample was diluted with gelatin buffer, and the total volume of the injected liquid was 200 μL. The mice were observed for 1 to 3 days, and the death of the mice was recorded. As a result, all the mice with 1MLD of natural toxin died, and the mice with three different injection doses of negative control group and recombinant t...

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Abstract

The invention provides a clostridium perfringens epsilon toxin mutant protein as well as a preparation method, application and a vaccine thereof, and relates to the technical field of biology. The clostridium perfringens epsilon toxin mutant protein includes alanine mutated by 151-position amino acid of clostridium perfringens epsilon toxin shown in an amino acid sequence SEQ ID NO.3. Compared with a wild type, the clostridium perfringens epsilon toxin protein has significantly reduced toxicity and good immunogenicity. The vaccine can alleviate technical problems such as complex production process, high cost, complex antigen, inter-batch difference and large side effects of inactivated vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Clostridium perfringens epsilon toxin mutant protein and its preparation method, application and vaccine. Background technique [0002] Clostridium perfringens (Clostridium Perfringens), also known as Clostridium welchii, is an important zoonotic disease. Clostridium perfringens can cause traumatic gas gangrene and human food poisoning, and can also cause cattle and sheep Necrotic enteritis, sheep fast disease, cattle and sheep enterotoxemia and lamb dysentery, therefore have a huge threat to animal husbandry. Clostridium perfringens mainly secretes four types of exotoxins, α, β, ε, and ι. According to the different types of exotoxins secreted, Clostridium perfringens can be divided into five types: A, B, C, D, and E. Toxic type. [0003] Clostridium perfringens has the characteristics of acute onset, short course of disease and high mortality, so people or animals are prone to s...

Claims

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Application Information

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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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