结核分枝杆菌基因Rv2660c、Rv2460c、Rv3875、Rv3804c细胞表位融合蛋白的表达及其免疫原性评价

ObjectiveTo analyse the immunogenicity of a fusion protein containing cell epitopes ofMycobacterium tuberculosisgenesRv2660c,Rv2460c,Rv3875 andRv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines.MethodsCell epitopes ofRv2660c,Rv2460c,Rv3875 andRv3804cwere fused in series to form a new antigen gene (namedmsv). Thenmsvwas cloned into the prokaryotic expression vectorpEASY-Blunt E1. The fusion protein msv was expressed bypEASY-Blunt E1 under the induction of isopropyl-β-d-thiogalactoside (IPTG). Purified the protein by affinity chromatography and identified the protein by SDS-PAGE and Western blot. To evaluate the immunogenicity of the protein, the mice were immunized with the purified fusion protein, and the titer of the antibody in mice serum was evaluated by ELISA. Besides, splenocytes of immunized mice were separated and splenocytes proliferation was determined under the stimulation of the protein.ResultsThe prokaryotic expression plasmid carryingmsvgene was constructed successfully and msv protein could be expressed by the plasmid under the induction of IPTG. SDS-PAGE and Western blot results confirmed that a purified protein (relative molecular mass was 41.3×10³) was obtained. ELISA result indicated that the titer of the antibody in msv immunized mice serum was about 1:81 920.The spleen lymphocyte proliferation assay showed that after immunization with msv protein, significant proliferation of antigen-sensitized lymphocytes was observed.ConclusionThe fusion protein msv was successfully expressed and purified, which can induce humoral and cellular immunity in mice. It may be used as an antigen component for the development of TB vaccine in the future.