Cloning of alpha-beta fusion gene from Clostridium perfringens and its expression

World J Gastroenterol. 2006 Feb 28;12(8):1229-34. doi: 10.3748/wjg.v12.i8.1229.

Abstract

Aim: To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression.

Methods: Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay.

Results: The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.

Conclusion: The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Toxins / genetics*
  • Bacterial Toxins / immunology
  • Bacterial Toxins / toxicity
  • Base Sequence
  • Blotting, Western
  • Calcium-Binding Proteins / genetics*
  • Calcium-Binding Proteins / immunology
  • Calcium-Binding Proteins / toxicity
  • Cloning, Molecular / methods*
  • Clostridium perfringens / genetics*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / genetics
  • Gene Expression
  • Gene Fusion
  • Genes, Bacterial
  • Genetic Vectors
  • Mice
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / physiology
  • Type C Phospholipases / genetics*
  • Type C Phospholipases / immunology
  • Type C Phospholipases / toxicity

Substances

  • Bacterial Toxins
  • CPB protein, Clostridium perfringens
  • Calcium-Binding Proteins
  • Recombinant Fusion Proteins
  • Type C Phospholipases
  • alpha toxin, Clostridium perfringens