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Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof

A Clostridium perfringens and co-expression carrier technology, applied in the field of bioengineering, can solve the problems of insufficient expression and inability to produce immune protection in immunized animals, so as to achieve good immunogenicity, facilitate large-scale production, and control quality Effect

Inactive Publication Date: 2014-11-05
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recombinant plasmid is a series of multiple toxin genes, and can express up to three toxins at the same time. When the expression is induced, the expression level is often insufficient, resulting in the inability of the immunized animals to produce sufficient immune protection.

Method used

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  • Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof
  • Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof
  • Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Clostridium perfringens α, β 1 , Β 2 Construction and expression of co-expression vector of ε and ε toxin

[0044] 1.α, ε, β 1 , Β 2 Amplification of toxin coding genes

[0045] Inoculate the purchased standard strains of C. perfringens C59-1 and C60-2 into freshly prepared anaerobic liver soup medium and cultivate overnight. 600 When it reaches 0.6-0.8, the anaerobic liver soup culture solution is collected aseptically, and the genomic DNA of C59-1 and C60-2 is extracted with the bacterial genome extraction kit as a PCR template to amplify each toxin gene.

[0046] PCR reaction system: 50μL MasterMix, 2μL upstream and downstream primers, 16μL template, 30μL deionized water; DNA gel recovery kit to recover four toxin coding genes;

[0047] 2.pETDuet-1-α and pRSFDuet-1-β 2 Vector construction

[0048] The two vectors pETDuet-1 and pRSFDuet-1 were digested with EcoRI and PstI. 40μL digestion system: 4μL of 10×H buffer, 1μL of EcoRI and PstI, 24μL of carrier, 8μL of deion...

Embodiment 2

[0055] Example 2: α, ε, β 1 , Β 2 Co-expression result detection of four toxins

[0056] 1 Preparation of SDS-PAGE solution:

[0057] Tris-glycine buffer: 25mmol / L Tris, 250mmol / L glycine (pH8.0), 0.1% SDS.

[0058] 2×SDS loading buffer: 100mmol / L Tris.C1 (pH8.0), 200m mol / L mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol.

[0059] Coomassie Brilliant Blue Staining Solution: Dissolve 0.25g of Coomassie Brilliant Blue R250 in 45ml methanol, 45ml water and 10ml glacial acetic acid.

[0060] Decolorizing solution: 30% methanol, 10% glacial acetic acid, distilled water to make up the volume to 100ml.

[0061] 2 SDS-PAGE experiment

[0062] (1) Wash the glass plate, fix it on the electrophoresis tank, and seal the edge with 2.0% agarose. Prepare 5ml of 15% separation glue according to the formula of molecular cloning, and quickly inject it between two glass plates, with the top of the glue covering 1ml of double distilled water. After the separation gel has solidified, pour out ...

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Abstract

The invention discloses a clostridium perfringens alpha, beta 1, beta 2 and epsilon toxin coexpression vector; vectors are respectively pETDuet-1 and pRSFDuet-1 expressed in the same host bacteria, and the vectors are respectively connected with clostridium perfringens alpha and epsilon toxin encoding genes and clostridium perfringens beta 1 and beta 2 toxin encoding genes. On the basis, the invention also discloses a construction method and an expression method of the coexpression vector.

Description

Technical field [0001] The invention relates to a multi-toxin co-expression vector obtained by using gene technology and a construction and expression method thereof, belonging to the field of bioengineering. Background technique [0002] The prevention and control of multiple serotypes of animal infectious diseases is one of the important reasons that currently plague veterinary scientists. Looking for the ability to express multiple proteins at the same time, and the immunogenicity of the protein is not destroyed and changed, and it can also affect multiple serotypes. Subunit vaccines for prevention and control are a technical problem that needs to be solved urgently in this field. [0003] Clostridium perfringens (clostridium perfringens), a gram-positive spore-forming anaerobe, is widely distributed in nature and can be found in soil, sewage, food, feces, etc. It is also a normal composition of human intestinal flora section. The pathogenic effect of this bacteria is mainly r...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66
Inventor 周继章韩广伟
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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