抗结核分枝杆菌DNA疫苗的构建及免疫原性研究

Construction and immunogenicity research on a therapeutic DNA vaccine of Mycobacterium Tuberculosis

目的:构建抗结核分枝杆菌(TB)DNA疫苗并进行体内外的鉴定。方法:采用PCR技术分别扩增TB(H37Rv)抗原单基因Ag85a、Ag85b以及融合基因Ag85a-Esat6和Ag85b-Esat6融合基因,其5'端均含人粒细胞-巨噬细胞集落刺激因子(GM-CSF)信号肽,将目的基因分别亚克隆于p VAX1真核表达载体,得到Ag85a/p VAX1、Ag85b/p VAX1、Ag85a-Esat6/p VAX1和Ag85b-Esat6/p VAX1四种质粒;经酶切和测序鉴定正确后,质粒转染COS-7细胞48 h,取上清液检测目的蛋白表达情况;利用在体电脉冲技术(EP)将质粒注射BALB/c小鼠双侧胫前肌,检测特异性的体液和细胞免疫应答。结果:四种质粒基因片段方向、序列正确;Western blot检测COS-7细胞转染48 h后的上清,均有清晰特异性的目的蛋白条带,并定量检测到Esat6蛋白的表达水平可达10 ng/ml;小鼠免疫实验结果表明,仅质粒Ag85b/p VAX1和Ag85a-Esat6/p VAX1可诱导针对结核抗原分泌高水平IFN-γ的Th1型免疫应答和特异性杀伤应答,其中通过ELISA检测小鼠血清的lg G和ELISPOT检测脾细胞分泌IFN-γ的实验结果阳性率均为100%,且Ag85a-Esat6/p VAX1的效果较好些,与卡介苗具有显著性差异。结论:成功构建了抗TB的DNA疫苗,其中Ag85a-Esat6/p VAX1质粒DNA疫苗的免疫效果较好,为进一步研发治疗性的TB DNA疫苗奠定基础。

Objective： To develop effective therapeutic Mycobacterium Tuberculosis（ TB） DNA vaccines and research their immune activity in vitro and in vivo. Methods： TB（ H37Rv） antigen genes Ag85 a,Ag85b,Ag85a-Esat6 and Ag85b-Esat6 fusion gene,whose 5＇ terminal have gene encoding human granulocyte colony stimulating factor（ GM-CSF）,were amplified by PCR. Then these genes were subcloned into eukaryotic vector p VAX1,which were respectively named Ag85 a / p VAX1,Ag85 b / p VAX1,Ag85a-Esat6 / p VAX1 and Ag85b-Esat6 / p VAX1. The new plasmids were analyzed by restriction digestion and DNA sequencing. The plasmids were transfected into COS-7 cells in vitro with Lipofecta mineTM2000. At 48 h after trasfection,the supernatants were analysised by Western Blot and Elisa. The plasmids were injected into BALB / c mice by electroporation in situ and specific humoral and cellular immune responses were exa mined. Results： The four plasmids were constructed correctly by genetic analysis. At 48 h after transfection,Ag85 a,Ag85b,Ag85aEsat6 and Ag85b-Esat6 genes expressed respectively,and objestive proteins were detected by Western blot clearly specially. It was up to10 ng / ml in supernatants detected by ELISA of Esat6. Experiment performed in BALB / c mice injected with only plasmids Ag85a-Esat6 / p VAX1 or Ag85 b / p VAX1 showed,protective antibodies of Ag85 a or Ag85 b were produced,and Ag85 a or Ag85 b specific killing response and Th1-type immune response with high level of IFN-γ were also induced. Positive ratios were both 100% in ELISA detecting anti-Ag85 a or anti-Ag85 b Ig G and ELISPOT detecting specific spleenocytes secreting IFN-γ. The plasmid Ag85a-Esat6 / p VAX1 had better immune efficacy than Ag85 b / p VAX1 and significant deference from BCG. Conclusion： The TB DNA vaccines were constructed successfully,and Ag85a-Esat6 / p VAX1 induced the most strong specific immune activity in vitro and in vivo.