HBx缺失突变型质粒的构建及在HepG2细胞中的表达.docVIP

PAGE PAGE 10 HBx缺失突变质粒的构建以及在HepG2细胞中对HBV前基因组RNA转录的影响 罗 莉1 郭进军1 雷宇2 罗娜1 龚倩1 何松1（400010 重庆，重庆医科大学附属第二医院消化内科1；400010 重庆，重庆医科大学病毒性肝炎研究所2） [摘要] 目的 构建HBx基因缺失突变型质粒pUC-HBV1.0.X7，研究 HBx对HBV前基因组RNA（pregenomic RNA，pgRNA）转录的影响。方法 以包含HBV全长基因组的野生型质粒pUC-HBV1.0为模板，运用PCR介导的定点突变的方法，将pUC-HBV1.0 的HBx读码框第7位氨基酸引入终止密码，构建HBx基因缺失突变的HBV复制质粒pUC-HBV1.0.X7，并用HindⅢ、SacI双酶切和DNA测序鉴定。然后经转染试剂PolyJet? Reagent介导，将质粒pUC-HBV1.0和pUC-HBV1.0.X7分别瞬时转染到HepG2细胞中。Western blot检测HBx蛋白的表达，real-time RCR相对定量分析HBV前基因组RNA( pgRNA)的转录。结果 经Hind Ⅲ、SacI双酶切和DNA测序证实，HBx基因缺失突变型质粒pUC-HBV1.0.X7构建成功，且Western blot尚未检测到HBx蛋白的表达。Real-time RCR结果显示在转染后48小时HBx突变组转录的pgRNA与野生组相比没有统计学差异（p 0.05），但是在转染后 96小时HBx突变组中转录的pgRNA减少50%-70% （p 0.05）。结论 HBx缺失突变型质粒构建成功，在HepG2细胞中HBx缺失能使HBV前基因组RNA的转录减少，HBx能上调HBV前基因组RNA的转录。 [关键词] HBV X蛋白；前基因组RNA；定点突变技术 [中图法分类号] R57 [文献标志码] A The construction of the HBx-defective mutant plasmid and the effect of HBx on HBV pregenomic RNA transcription in HepG2 cells Luo Li1 Guo Jinjun1 Lei Yu2 Luo Na1 Gong Qian1 He Song1 (1Department of Gastroenterology, The 2nd Affiliated Hospital of Chongqing Medical University, Chongqing, [基金项目] 国家自然科学基金 [通信作者] 何松，E-mail: hedoctor@ 400010; 2Institute for Viral Hepatitis, Chongqing Medical University, Chongqing, 400010, China) [Abstract] Objective To construct the HBx-defective muant plasmid pUC-HBV1.0.X7, and investigate the effect of HBx on HBV pregenomic RNA（pgRNA）transcription. Methods The wild-type plasmid pUC-HBV1.0 which containing full-length HBV genome was used as a template, and the plasmid pUC-HBV1.0.X7 with a stop codon at amino acid 7 of HBx was constructed by site-directed mutagenesis. The plasmid pUC-HBV1.0.X7 was identified by HindⅢ and SacI restriction digestion and DNA sequencing. Then the plasmids pUC-HBV1.0 and pUC-HBV1.0.X7 were separately transfected into HepG2 cells with PolyJet? Reagent. The expression of HBx was detected by Western blot and the transcription of HBV pgRNA was relatively quantified by real-time RCR. Res