Construction of the prokaryotic expression vector with Rv1246c and Rv1247c genes of Mycobacterium tuberculosis and its expression in E. coli

To analyze the structures of two proteins encoded by the Rv1246c and Rv1247c gene of Mycobacterium tuberculosis by bioinformation with Bioedit,Dnaman and Pfam;these gene were amplified by PCR from genome of M.tuberculosis H37Rv and inserted into prokaryotic expression vector pET32a(+),then the recombinant plasmids were transformed into(E.coli) BL21(DE3) strain and induced by IPTG.The expressed recombinant proteins were identified by SDS-PAGE and Western-blot and the recombinant proteins were purified by cutting the gels down and reclaiming.The polyclonal antibodies were prepared to immunizing the New Zealand rabbits with the purified proteins,and the specific IgG antibodies titer were determinded by indirect ELISA.Results showed that the protein encoded by Rv1246c and Rv1247c shared similarl motifs with the family of RelBE in E.coli.The homologies of Rv1246c protein with RelE of E.coli and Rv1247C protein were 21.55% and(23.85%) respectively.These two genes were amplified by PCR and were subcloned to pET32a(+).The fused proteins were successfully expressed in E.coli BL21 and had relative molecular mass of 31kD and 29kD respectively.The fused proteins were evaluated by Western-blot method and the specific antibodies titer in the serum of rabbits were analyzed by indirect ELISA.It was fuun that the titer was greater than 1∶40 000.It is concluded that the recombinant expression plasmids pET-Rv1246c and pET-Rv1247c are successfully constructed firstly and the fusion proteins are obtained from the induced E.coli BL21.These result provided the basis for the further study of the gene Rv1246c and Rv1247c.