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

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.

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β 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℃, 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 α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.