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

摘要

AIM:To study the cloning of α-β fusion gene from Clos-tridium perfringens and the immunogenicity of α-β fusionexpression.METHODS:Cloning was accomplished after PCR amplifi-cation from strains NCTC64609 and C58-1 of the protec-tive antigen genes of α-toxin and β-toxin.The fragmentof the gene was cloned using plasmid pZCPAB.Thisfragment coded for the gene with the stable expressionof α-β fusion gene binding.In order to verify the exactlocation of the α-β fusion gene,domain plasmids wereconstructed.The two genes were fused into expressionvector pBV221.The expressed α-β fusion protein wasidentified by ELISA,SDS-PAGE,Western blotting andneutralization assay.RESULTS:The protective α-toxin gene(cpa906)andthe β-toxin gene(cpb930)were obtained.The recombi-nant plasmid pZCPAB carrying α-β fusion gene was con-structed and transformed into BL21(DE3).The recombi-nant strain BL21(DE3)(pZCPAB)was obtained.After therecombinant strain BL21(DE3)(pZCPAB)was induced by42℃,its expressed product was about 22.14% of totalcellular protein at SDS-PAGE and thin-layer gel scanninganalysis.Neutralization assay indicated that the antibodyinduced by immunization with α-β fusion protein couldneutralize the toxicity of α-toxin and β-toxin.CONCLUSION:The obtained α-toxin and β-toxin genesare correct.The recombinant strain BL21(DE3)(pZCPAB)could produce α-β fusion protein.This protein can beused for immunization and is immunogenic.The anti-body induced by immunization with α-β fusion proteincould neutralize the toxicity of α-toxin and β-toxin.