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目的 利用T7RNA聚合酶介导的胞质表达系统增加目的抗原的表达量 ,提高基因疫苗的免疫效果。方法 (1)构建带有T7启动子、脑心肌炎病毒 (EMCV) 5′非编码区和柯萨奇B组病毒衣壳蛋白VP1的质粒pT7EMCVP1。将该质粒和编码T7RNA酶的pAR 3132共转染HeLa细胞和小鼠腹腔巨噬细胞 ;(2 )将上述质粒分别转化减毒鼠伤寒沙门菌SL72 0 7,让带有不同质粒的两种载体细菌共感染小鼠腹腔巨噬细胞。结果 (1)经共转染 ,在HeLa细胞和小鼠腹腔巨噬细胞中 ,目的抗原VP1在胞质中的表达比单独转染真核表达质粒pcDNA3VP1增加 2～ 4倍 ;(2 )两种质粒载体细菌感染小鼠巨噬细胞后 ,目的抗原VP1也能在细胞中较好表达。结论 pT7EMCVP1和pAR 3132能在HeLa细胞和小鼠腹腔巨噬细胞胞质中表达 ,且表达量比单独转染真核表达质粒pcDNA3VP1明显增加
OBJECTIVE: To improve the immunogenicity of gene vaccines by using T7 RNA polymerase-mediated cytoplasmic expression system to increase the expression of antigen of interest. Methods (1) Construction of plasmid pT7EMCVP1 harboring the T7 promoter, 5 ’non-coding region of encephalomyocarditis virus (EMCV) and VP1 of coxsackie B virus capsid protein. The plasmid and pAR 3132 encoding T7 RNase were co-transfected into HeLa cells and mouse peritoneal macrophages; (2) The above plasmids were respectively transformed into attenuated Salmonella typhimurium SL72 0 7, and two vectors carrying different plasmids Bacteria infect murine peritoneal macrophages. Results (1) The co-transfection of HeLa cells and mouse peritoneal macrophages, the expression of the target antigen VP1 in the cytoplasm compared with the eukaryotic expression plasmid pcDNA3VP1 transfection alone increased 2 to 4 times; (2) two Plasmid vector-infected mouse macrophages, the target antigen VP1 can also be better expressed in the cells. Conclusion pT7EMCVP1 and pAR 3132 can be expressed in the cytoplasm of HeLa cells and mouse peritoneal macrophages, and the expression levels of pT7EMCVP1 and pAR 3132 are significantly higher than that of pcDNA3VP1 transfected with eukaryotic expression plasmid alone