Analysis of immunogenicity of tuberculosis fusion protein consisting of Ag85B, Mpt64 and HspX antigens expressed in replication and dormancy bacilli

Objective: To construct protective immunity to Mycobacterium tuberculosis latent infection, a novel fusion protein consisting of HspX, the 190 to 198 peptide of Mpt64 and Ag85B, which were confirmed to be the effective protective antigens mainly expressed in the dormancy and exponential phase of growth, was constructed and its immunogenicity was investigated. Methods: Ag85B and Mpt64 _190-198-HspX sequences were amplified by PGR and cloned into plasmids pET-28a. The fusion protein, Ag85B-Mpt64 _190-498-HspX (AMH) was expressed in E. coli BL21 and purified with Ni-NTA resins. C57BL/6 mice were immunized three times at 2-week intervals subcutaneously with AMH formulated with the adjuvant composed of dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). Humoral and cell-mediated immunity responses were analyzed at five weeks after the last injection. Results: AMH was expressed stably in E. coli and could be purified well by Ni-NTA affinity chromatography. C57BL/6 mice immunized with AMH subunit vaccine generated specific cellular and humoral immunologic response to the stimulation of Ag85B, Mpt64 _190-198 and HspX. Conclusion: It suggested that AMH was a promising candidate antigen of tuberculosis subunit vaccine.