Cloning of α-β fusion gene from Clostridium perfringens and its expression

AIM: To study the cloning of a-beta fusion gene from Clos-tridiumpel-fringens and the immunogenicity of a-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 a-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 degrees C, 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 a-toxin and p-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce a-p fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with a-P fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin. (c) 2006 The WJG Press. All rights reserved.