[Immune regulation of T-bet adjuvant on Ag85B DNA vaccine against Mycobacterium tuberculosis].

Aim: To construct a novel Ag85B DNA vaccine based on the genetic adjuvant of T-bet known as Th1 transcription factor and to research the immunoregulation function of the DNA complex vaccine.

Methods: Ag85B gene and T-bet gene were amplified by RT-PCR, and cloned into pcDNA3.1 plasmid to construct recombinant plasmids pcDNA3-Ag85B and pcDNA3-T-bet, respectively. The recombinant plasmids were transfected into RAW264.7 cells using Lipofectamine(TM); 2000 reagent to detect the expressions of Ag85B and T-bet proteins by Western blotting. BALB/c mice were immunized by three intramuscular inoculations with pcDNA3.1-FLAG-T-bet in combination with pcDNA3.1-FLAG-Ag85B. Two weeks after the last immunization, the anti-Ag85B antibody titres in sera were tested by ELISA. Meanwhile, spleen lymphocyte suspension was cultured in the context of Ag85B, and then the secretion of cytokines in the culture fluid was tested by ELISA.

Results: Plasmid proteins were successfully expressed in a dose-dependent manner. Additionally, T-bet/Ag85B complex not only induced obviously higher IgG2a titre with the lower IgG1, but also stimulated the increased secretion of IFN-γ and IL-2 with the concomitant repression of IL-4 and IL-10.

Conclusion: T-bet can enhance Ag85B-specific IgG2a antibody response and convert T cell subsets to a Th1-predominant immune response.