为研究针对结核分枝杆菌潜伏感染的DNA疫苗，基于质粒A39构建了p-VAX1-Ag85B-Rv3425-Rv2029c-PPE26 (V569)质粒DNA，并对其免疫原性及保护性进行初步研究。免疫性评价试验共分6组：PBS、p-VAX1-Ag85B(A)、p-VAX1-Ag85B-Rv3425(A3)、A39、V569和BCG，采用左后腿肌内注射C57BL/6小鼠，用流式细胞术和酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)分别检测细胞免疫和体液免疫水平；构建斑马鱼-海分枝杆菌潜伏感染模型，将PBS、A、A3、A39、BCG、V569分别通过腹腔注射免疫斑马鱼后，每日注射地塞米松10ug诱导海分枝杆菌复发感染，对斑马鱼肝脏进行菌落计数并绘制生存曲线。结果显示，与BCG组相比，V569能引发实验小鼠强烈的细胞免疫反应(IFN-γ高水平分泌)，外周血CD4^＋/CD8^＋ T细胞比例明显增加。在斑马鱼-海分枝杆菌潜伏感染复发模型中，与BCG 免疫组相比，V569免疫斑马鱼后可显著减少其肝脏中海分枝杆菌数量，斑马鱼存活情况得到显著改善，表明V569 DNA疫苗可能是一种抗结核潜伏感染的候选DNA疫苗。

We constructed the DNA vaccine Ag85B-Rv3425 -Rv2029c-PPE26 (V569) plasmid based on p-VAX1-Ag85B-Rv3425-Rv2029c (A39) and evaluated its immunogenicity in mice. The levels of cellular immunity and humoral immunity against PBS, BCG, p-VAX1-Ag85B (A), p-VAX1-Ag85B-Rv3425 (A3), A39 and V569 were detected by enzyme-linked immunosorbent assay (ELISA) and flow cytometry after vaccination in mice. We found that V569 could trigger stronger Th1 immune response by augmenting the secretion of IFN-γ and significantly improve the CD4^＋/CD8^＋ T cell ratio in immunized mice compared to BCG. In addition, we evaluated the V569 as post-exposure DNA vaccine and found that it reduced bacterial burdens upon reactivation compared to BCG. The protection of V569 against latent TB infection was determined by zebrafish-Mycobacterium marinum latent infection model. In conclusion, V569 DNA vaccine may be a potential candidate DNA vaccine against latent tuberculosis infection.