Construction of recombinant adenovirus expressing Ag85B of Mycobacterium bovis and its cellular immunoproperties in mice

Abstract:

Abstract: [Objective] We constructed recombinant adenovirus rAd-Ag85B expressing antigen 85B of Mycobacterium bovis and analyzed cellular immune responses after mice immunization. [Methods] We amplified the fbpB gene from genomic DNA of BCG by PCR and cloned into pMD18-T vector for sequence analysis. Then we constructed pDC516-Ag85B and co-transfected with plasmid pBHGfrtΔE1,3FLP into Ad293 cells by lipofectamineTM2000. Then we identified the recombinant Ad-Ag85B virus by RT-PCR, Western blot assay and TEM observation after negative staining. BALB/c mice were subcutaneously immunized with the dose of 108 TCID50 rAd-Ag85B or rAd. Two weeks after immunization, CD69 expression, T lymphocyte proliferation and ELISPOT were detected in spleen cells of immunized mice. [Results] We obtained the correct recombinant adenovirus rAd-Ag85B expressed Ag85B antigen. The level of both splenic CD69+CD4+T and CD69+CD8+T cells of rAd-Ag85B group were significantly higher than those of rAd control. The SI index of rAd-Ag85B was much higher than rAd. In ELISPOT assay more IFN-γ specific secreting cell spots other than IL-4 were detected in rAd-Ag85B group. [Conclusion] The recombinant adenovirus rAd-Ag85B can induce specific Th1 cell immune responses in mice.