独立表达Ag85A与ESAT6的抗结核DNA疫苗的构建  

The construction of Ag85A and ESAT6 co-expression eukaryotic plasmid

作　　者：张雷[1] 杨小康[1] 胡东[1] 吴静[1] 王文洋[1] 杜久伟[1] 许礼发[1] 张荣波[1] 

机构地区：[1]安徽理工大学医学院免疫与检验教研室,安徽淮南232001

出　　处：《广东医学》2013年第20期3102-3104,共3页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81202294;81172778;61170172);安徽省自然科学基金资助项目(编号:1208085QH162);安徽省淮南市科技计划项目(编号:2011A07915;2012A00912)

摘　　要：目的以pIRES为载体建立可同时独立表达Ag85A与ESAT6两种抗原的抗结核二价疫苗,探究疫苗剂量对质粒基因体外表达水平的影响。方法 PCR扩增获得Ag85A基因和ESAT6基因,分别插入pIRES质粒的多克隆位点A和B,构建pIRES-Ag85A-ESAT6重组质粒。脂质体2000将重组质粒转入RAW264.7细胞系,Western blot法检测目的蛋白的表达水平。结果成功构建pIRES-Ag85A-ESAT6重组质粒,在转染后的RAW264.7细胞同时高表达Ag85A与ESAT6,其表达水平与转染的剂量呈正相关。结论 pIRES-Ag85A-ESAT6二价疫苗的构建将为基于多个抗原同时独立表达的新型多价疫苗设计提供了思路。Objective To construct a bivalent antituberculosis vaccine, which expressed both Ag85A and ESAT6 antigen independently, and to explored the impact on the expression of plasmid gene of different levels of vaccine in vitro. Methods The Ag85A and ESAT6 open - reading frames were amplified by RT - PCR, and cloned into the multiple cloning sites A （MCSA） and B （MCSB） of pIRES vector, respectively. The recombinant plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent. The Ag85B and ESAT6 expression was measured in transfected cells by Western blot assay. Results The pIRES -Ag85A -ESAT6 plasmid was successfully constructed with co -expressing of two antigens independently in a dose - dependent manner. Conclusion The recombinant plasmid pIRES - Ag85A - ESAT6 serves as a basis for further research of multiple - valent vaccines to prevent tuberculosis.

关 键 词：多价疫苗 结核分枝杆菌 AG85A ESAT6 

分 类 号：R378.911[医药卫生—病原生物学]