共表达ag85a与esat6真核表达质粒的构建.docVIP

独立表达Ag85A与ESAT6的构建 张雷1，杨小康1，胡东12，吴静12, *，王文洋1，陈丽萍1，许礼发12, *，张荣波12, * （安徽理工大学：1医学院免疫与检验教研室；2免疫与感染研究所，安徽 淮南232001） 【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MCSA和MCSB，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：pIRES-Ag85A-ESAT6重组质粒，在转染后的RAW264.7细胞表达，表达水平与转染的剂量呈正相关。结论：pIRES-Ag85A-ESAT6疫苗。 【关健词】：疫苗，结核分枝杆菌，Ag85A，ESAT6 Construction of enkaryotic expression plasmid co-expressing Ag85A and ESAT6 Lei Zhang1, Xiao-kang Yang1, Dong Hu1, 2, Jing Wu1, 2, *, Wen-yang Wang1,, Li-ping Chen1,, Li-fa Xu1, 2,*, Rong-bo Zhang1, 2, * 1 Department of Medical Immunology and Test, Medical School, Anhui University of Science and Technology; 2 Institute of Infection and Immunology, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China * Correspondence to: Professor. Zhang Rong-bo (Email: lory456@126.com), Vice Professor. Li-fa Xu (Email: lfxu2003@) and Vice Professor. Wu Jing (Email: wujing8008@126.com). No. 25 Middle Dongshan Road, Huainan, Anhui 232001, P.R. China ABSTRACT Objective: To construct an antituberculosis bivalent vaccine which can express both Ag85A and ESAT6 antigen independently, then explored the impact of the expression of the plasmid gene in vitro with different levels of vaccine. Methods: The Ag85A and ESAT6 open-reading frame was amplified by RT-PCR, and cloned into the multiple cloning site A and B of pIRES vector, respectively. The recombinant plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent. The high level Ag85B and ESAT6 were detected in transfected cells by western-blot assay. Results: The pIRES-Ag85A-ESAT6 construct is available for co-expressing two antigens independently, and it expressed in a dose-dependent manner. Conclusions: the recombinant plasmid of pIRES-Ag85A-ESAT6 serve as a basis for further research of multiple vaccines to prevent tuberculosis Key words: multiple vaccines, Mycobacterium tuberculosis,