Prokaryotic Expression and Immunogenicity Analysis of a Fusion Protein Containing Cell Epitopes of Mycobacterium tuberculosis Rv2660c, Rv2460c, Rv3875 and Rv3804c Genes

Abstract

To analyse the immunogenicity of a fusion protein containing cell epitopes of Mycobacterium tuberculosis genes Rv2660c, Rv2460c, Rv3875 and Rv3804, and to evaluate the feasibility of using it as a novel target antigen for developing multi-stage TB vaccines.  Methods  Cell epitopes of Rv2660c, Rv2460c, Rv3875 and Rv3804c were fused in series to form a new antigen gene (named msv). Then msv was cloned into the prokaryotic expression vector pEASY-Blunt E1. The fusion protein msv was expressed by pEASY-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.  Results  The prokaryotic expression plasmid carrying msv gene 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.  Conclusion  The 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.

Keywords: Mycobacterium tuberculosis, Multi-stage, Rv2660c, Rv2460c, Rv3875, Rv3804c, Epitope