CAJ | 학술논문

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pVAX1／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。
선취결핵분지간균잠복상관항원Rv2029c、결핵분지간균우수항원Ag85A화Rv3425，구건침대잠복감염적결핵분지간균DNA역묘pVAX1／Ag85A-Rv3425-Rv2029c （A39），병대기면역원성진행연구。수선용취합매련반응（PCR）확증Ag85A기인，구건중조질립pVAX1／Ag85A （A）；PCR확증 Ag85A-Rv3425련접편단，삽입pVAX1재체，구건중조질립pVAX1／Ag85A-Rv3425（A3）；PCR확증Rv2029c기인，삽입A3，구건중조질립pVAX1／Ag85A-Rv3425-Rv2029c （A39）。장구건성공적중조질립전입 HEK293T세포，단백면역인적법험증질립재진핵세포중득도표체。재대장애희균BL21중성공표체화순화거제신호태적Ag85A、Rv3425화Rv2029c단백。용질립면역C57BL／6소서，공분위5조：PBS、pVAX1、A、A3화A39조，채용전맥충도입면역，매2주면역1차，공3차，용매련면역반점검측（ELISPOT）、매련면역흡부시험（ELISA）、류식세포술등방법검측세포면역화체액면역수평。결과현시，A39면역소서후，능인발강렬적세포면역반응﹝γ간우소（IFN-γ）、종류배사인자α（TNF-α）화백세포개소2（IL-2）고수평분비﹞，외주혈CD4＋／CD8＋ T세포비치증가，CD8＋천공소＋ T세포비례증가。결과표명，구건적A39 DNA역묘능인발강렬적면역반응，현시출량호적항결핵잠력，가작위결핵분지간균신형후선역묘。
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 pVAX 1 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-α,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 .