Abstract: Objective To construct DNA vaccine of dormancy related antigen Rv1733c of Mycobacterium tuberculosis (Mtb), and to evaluate its immunogenicity in mice.  Methods Rv1733c gene was cloned into the eukaryotic expression vector pcDNA 3.1(-) from the plamid pMD-18T-Rv1733c, which was constructed previously and preserved in our lab. The constructed plasmid was named pcDNA-Rv1733c. P815 cells were stably transfected with the plasmid pcDNA-Rv1733c, and were detected the protein expression of Rv1733c by indirect immunofluorescence(IFT). The mice BALB/c were divided randomly into three groups named pcDNA-Rv1733c DNA, saline and BCG, 10 mice per group. The mice were immunized with pcDNA-Rv1733c DNA or saline intramuscularly 3 times at an interval of 2 weeks. BCG was immunized subcutaneously only once. The antigen specific antibody level and IgG2a/IgG1 subtype ratio in immunized mice were detected by ELISA every 2 weeks. At 8 weeks after the first immunization, the specific proliferation of spleno-lymphocytes of mice was detected by MTS ［3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium, inner salt］ method, and IFN-γ secreted by spleno-lymphocytes was detected by ELISPOT. The percentages of CD4⁺ and CD8⁺ T cells in spleno-lymphocytes were analyzed by flow cytometry, and the function of cytotoxicity T lymphocyte (CTL) was detected by lactate dehydrogenase(LDH) assay． Results The eukaryotic expression plasmid of Rv1733c gene was successfully constructed, named pcDNA-Rv1733c. In P815 cells stably transfected with pcDNA-Rv1733c plasmid，the expression protein of Rv1733c could be stably detected by IFT. In immunized mice, the pcDNA-Rv1733c plasmid could stimulate the production of the antigen specific antibody, and the antibody subtype biased to IgG2a，but with the extension of immunization time，the ratio of IgG2a/IgG1 was tended to balance. In pcDNA-Rv1733c DNA group, the stimulation index of spleno-lymphocytes was (2.00±0.36), and the cell number of secreted IFN-γ was (41.48±5.30) SFC/10⁶, which were both statistically higher than those in saline group (t=3.096,P<0.05). However, the percentages of CD4⁺ and CD8⁺ T cells ［(18.15±2.30)% and (7.68±1.34)%］, and the activity of CTL ［(29.52±1.96)%］ were not significantly different with those in saline group(t=0.571, P>0.05).  Conclusion The eukaryotic expression plasmid of Rv1733c was constructed successfully. The pcDNA-Rv1733c plasmid DNA could induce specific humoral and cellular immunity in mice. It may be used as the new TB vaccine against latent Mtb infection.

Key words: Mycobacterium tuberculosis, Antigens, bacterial, Vaccines, DNA, Antibody formation, Immunity, cellular