基因功能的研究是目前分子生物学中的热点和研究-第三军医大学学报.docVIP

HBV X基因转染L02细胞及其蛋白产物对HLA-DR表达的影响 张 卿，李俊刚，刘玉元，赵 博，张绪清 （400038 重庆，第三军医大学西南医院全军感染病研究所） [摘要] 目的 探讨脂质体和电穿孔法在HBV X基因转染L02细胞中的差别及X基因蛋白产物对HLA-DR表达的影响。方法 构建HBV X基因重组表达质粒，通过电穿孔和脂质体法转染人正常肝细胞L02细胞，以绿色荧光蛋白（enhance green fluorescent protein ，EGFP）表达水平评价转染效率，流式细胞技术检测L02细胞HBV X蛋白的表达水平。RT-PCR和流式细胞技术分析L02细胞、转染空载体以及转染HBV X基因重组表达质粒的L02细胞HLA-DR的表达差别。结果 采用阳离子脂质体Lipofectamine 2000的转染效率为（6.4±3.5）%，显著低于电穿孔法（41.46±5.8）%，P0.01。脂质体法转染的L02细胞HBV X蛋白表达水平为(5.1±3.9) %，显著低于电穿孔方法(37.5±4.1)%表达，P0.01。转染了HBV X基因重组表达质粒的L02细胞检测到HLA-DR mRNA和蛋白表达，而L02细胞本身及转染空载体的L02细胞未检测到HLA-DR的表达。 结论 HBV X基因真核表达载体pHBx-IRES2 -EGFP通过电穿孔法转染L02细胞较脂质体法有明显较高的转染效率和更高的目的基因的表达，HBV X基因诱导了L02细胞中HLA-DR的表达。 [关键词] HBV X基因；脂质体；电穿孔；HLA-DR [中图法分类号] [文献标志码] A Transfection of HBV X gene into L02 cell lines and the effect of X gene product on HLA-DR expression Zhang Qing, Li Jungang, Liu Yuyuan, Zhao Bo, Zhang Xuqing（Department of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, 400038 , China. ） [Abstract] Objective To investigate the efficiency differences between lipofectamine and electroporation on the transfection of HBV X gene into L02 cell lines and the effect of HBV X protein on HLA-DR expression. Methods pHBx-IES2-EGFP plasmid was constructed and transfected into L02 cell lines by the methods of lipofectamine and electroporation. Transfection efficiency was evaluated using the EGFP expression by fluorescence microscope. Flow cytometry were used to analyze HBx protein expression. RT-PCR and flow cytometry were used to analyze HLA-DR expression in L02 cell lines、L02 cell lines transfected with pIES2-EGFP and L02 cell lines transfected with pHBx-IES2-EGFP. Results Transfection efficiency of lipofectamine was (6.4±3.5) %, which was significantly lower than that of electroporation (41.46±5.8) %, P0.01. On the expression of HBx, the method of lipofectamine (5.1±3.9) % was significan