ABSTRACT
Objective To construct a recombinant Bacillus Calmette-Guerin ( BCG ) vaccine strain, rBCG::Rv3478-pMV261, expressing the Rv3478 protein of Mycobacterium tuberculosis and to inves-tigate its immunogenicity.Methods The gene fragments encoding Rv3478 antigen were amplified by PCR and then respectively cloned into pMV261 and pET-28a vectors to construct the recombinant expression plas-mids (Rv3478-pMV261 and Rv3478-pET-28a).The Rv3478-pMV261 plasmids were transformed into the BCG cells to construct the rBCG vaccine strains, while the Rv3478-pET-28a plasmids were transformed into Escherichia coli BL21 strains for the expression of Rv3478 protein.Polyclonal antibodies were induced in mice upon the immunization with Rv3478 protein.The rBCG vaccine strains overexpressing Rv3478 protein were screened out with Western blot assay.The C57BL/6 mice were divided into four groups including the PBS treated group, BCG treated group, rBCG::pMV261 ( R0) treated group and rBCG::Rv3478-pMV261 ( R3) treated group.All mice were sacrificed in 4 or 12 weeks after immunization.Enzyme-linked immunos-pot assay ( ELISPOT) , ELISA and flow cytometry analysis were performed to evaluate the induced humoral and cell-mediated immune responses in mice.Results The Rv3478 protein was successfully expressed and could induce polyclonal antibodies in mice.High levels of IFN-γand TNF-αwere detected in mice treated with R3, indicating that the immunization with R3 enhanced the cellular immunity.Moreover, the ratios of CD4+to CD8+T cells and the percentages of CD44+CD62L+T cells were increased in mice upon the immuni-zation with R3.Conclusion The recombinant BCG vaccine strain overexpressing Rv3478 protein could in-duce stronger cell-mediated immune responses in mice.It might be have a great significance as a new tuber-culosis( TB) vaccine strain against TB infection in the future.