针对潜伏结核感染的A39 DNA疫苗构建及其免疫原性研究

Construction and immunogenicity of A39 DNA vaccine against latent tuberculosis infection

选取结核分枝杆菌潜伏相关抗原Rv2029c、结核分枝杆菌优秀抗原Ag85A和Rv3425,构建针对潜伏感染的结核分枝杆菌DNA疫苗pVAXI/Ag85A-Rv3425-Rv2029c(A39),并对其免疫原性进行研究。首先用聚合酶链反应(PCR)扩增Ag85A基因,构建重组质粒pVAX1/Ag85A(A);PCR扩增Ag85A-Rv3425连接片段,插入pVAX1载体,构建重组质粒pVAX1/Ag85A-Rv3425(A3);PCR扩增Rv2029c基因,插入A3,构建重组质粒pVAX1/Ag85A-Rv3425-Rv2029c(A39)。将构建成功的重组质粒转入HEK293T细胞,蛋白免疫印迹法验证质粒在真核细胞中得到表达。在大肠埃希菌BL21中成功表达和纯化去除信号肽的Ag85A、Rv3425和Rv2029c蛋白。用质粒免疫C57BL/6小鼠,共分为5组:PBS、pVAX1、A、A3和A39组,采用电脉冲导入免疫,每2周免疫1次,共3次,用酶联免疫斑点检测(ELISPOT)、酶联免疫吸附试验(ELISA)、流式细胞术等方法检测细胞免疫和体液免疫水平。结果显示,A39免疫小鼠后,能引发强烈的细胞免疫反应[γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)和白细胞介素2(IL-2)高水平分泌],外周血CD4^+/CD8^+T细胞比值增加,CD8^+穿孔素^+T细胞比例增加。结果表明,构建的A39 DNA疫苗能引发强烈的免疫反应,显示出良好的抗结核潜力,可作为结核分枝杆菌新型候选疫苗。

The Mycobacterium tuberculosis DNA vaccine pVAX1/Ag85A-Rv3425-Rv2029c （A39） plasmid was constructed and its immunogenicity was then investigated. Ag85A gene was firstly amplified by polymerase chain reaction （PCR） and then cloned into pVAX1 to construct the recombinant plasmid pVAX1/Ag85A （A）. Ag85A-Rv3425 gene segment was amplified by PCR and then cloned into pVAX1 to construct the recombinant plasmid pVAX1/Ag85A-Rv3425 （A3）. Rv2029c gene was amplified by PCR and then cloned into plasmid A3 to construct the recombinant plasmid A39. HEK293T cells were transfected with the recombinant plasmids and the protein expression was detected by Western blotting. The proteins Ag85A, Rv3425 and Rv2029c were expressed in Escherichia coli BL21. C57BL/6 mice were divided into five groups named phosphate buffered saline （PBS）, pVAX1, A, A3 and A39. The mice were immunized with plasmids mediated by in vivo electroporation （EP） 3 times at an interval of 2 weeks. The levels of cellular immunity and humoral immunity were detected by enzyme-linked immunospot assay （ELISPOT）, enzyme-linked immunosorbent assay （ELISA） and flow cytometry. In the immunized mice, A39 could cause stronger cellular immunity： the high-level secretion of IFN-γ, IL-2 and TNF-a, and the increase in the proportion of CD4^＋/CD8^＋ T cells and CD8^＋ perforin^＋ T cells. Therefore, the constructed A39 DNA vaccine could induce stronger cellular immunity in mice and it may be used as a new tuberculosis vaccine against latent tuberculosis infection.