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.
出处
《广东医学》
CAS
CSCD
北大核心
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)
