论文部分内容阅读
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 Clos-tridium perfringens and the immunogenicity of α-β fusion expression. 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 fragment of the gene was cloned using plasmid pZCPAB.This fragmented 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 wereconstructed.The two genes were fused into expression vector pBV221.The expressed α-β fusion protein wasidentified by ELISA, SDS-PAGE, Western blotting andneutralization assay .RESULTS: The protectiveα-toxin gene (cpa906) andtheβ-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) (pZCPAB) was induced by42 ° C, its expressed p roduct 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-β fusion protein could be neutralize 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 be used 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.