Fusion expression of MPB70,MPB83,CFP-10 and ESAT-6 antigens from Mycobacterium bovis and analysis of their related properties

To investigate the fusion expression of MPB70,MPB93,CFP-10 and ESAT-6 antigens from Mycobacterium bovis and to analyze their related properties,the DNA fragments of 4 target genes mpb70,mpb83,cfp-10 and esat-6 were amplified from genomic DNA of M bovis isolates.The fusion genes cfp-10-esat-6 and mpb83-cfp-10-esat-6 were obtained through the splicing of overlapping extension technique.The gene mpb70 and fusion gene mpb83-cfp10-esat-6 were first inserted to the plasmid pUC-19 linker,and then the fusion gene mpb83-cfp-10-esat-6 were inserted to the expression plasmid pET32a(+),thus to obtain the resultant recombinant plasmid pET70-83-C10-E6.The fusion protein was expressed by transformation of pET70-83-C10-E6 into BL21(DE3)cells after IPTG induction and the recombinant soluble protein was purified with Ni2+ affinity chromatography.As demonstrated by ELISA assay,the expressed protein could be recognized by positive antiserum to M bovia and the mono-specific antisera against MPB70,MPB83,CFP-10 and ESAT-6 antigens.As demonstrated by Western blotting,this protein could be identified only by antiserum against M bovis,but not by antisera against M.paratuberculosis,Brucella,Babesia and infectious bovine rhinotracheitis virus.Heat treatment test showed that the fusion protein was heat-stable at 60℃ for one hour.It is concluded that 4 specific antigenic fusion proteins are obtained through fusion expression of MPB70,MPB83,CFP-10 and ESAT-6 antigens from M.bovis,and they can be used as the diagnostic antigen to detect the presence of the related infection.