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目的构建人载脂蛋白B mRNA编辑酶催化多肽样蛋白3G(APOBEC3G)真核表达载体,并探讨其抗乙型肝炎病毒(HBV)复制的作用。方法采用RT-PCR法从健康人外周血单个核细胞中克隆APOBEC3G基因编码区片段,构建pcDNA3.1-A3G真核表达载体,利用脂质体转染法将pcDNA3.1-A3G转染HepG2.2.15细胞,分别于转染后第24、48、72 h收集细胞培养上清液和细胞总蛋白,Western blot检测HBx蛋白表达情况,ELISA法检测细胞上清液中HBsAg和HBeAg表达情况。结果测序结果显示pcDNA3.1-A3G真核表达载体中APOBEC3G编码区序列存在1处碱基同义突变;HepG2.2.15细胞经APOBEC3G蛋白干扰后,HBx、HBsAg和HBeAg表达量逐渐降低,于72h表达量显著降低。结论成功构建了APOBEC3G真核表达载体pcDNA3.1-A3G;APOBEC3G在体外可以抑制HBV复制,为深入研究APOBEC3G作为抗HBV药物提供了实验基础。
OBJECTIVE: To construct an eukaryotic expression vector encoding APOBEC3G encoding human apolipoprotein B mRNA and explore its anti-HBV replication effect. Methods The coding region of APOBEC3G gene was cloned from healthy human peripheral blood mononuclear cells by RT-PCR. The eukaryotic expression vector pcDNA3.1-A3G was constructed and pcDNA3.1-A3G was transfected into HepG2 by lipofection. 2.15 cells were collected at 24,48,72 h after transfection were collected cell culture supernatant and total cellular protein, Western blot detection of HBx protein expression, ELISA assay of cell supernatant HBsAg and HBeAg expression. Results Sequencing results showed that there was one base synonymous mutation in APOBEC3G coding region in pcDNA3.1-A3G eukaryotic expression vector. The expression levels of HBx, HBsAg and HBeAg in HepG2.2.15 cells were decreased after APOBEC3G protein interference and expressed at 72h Significantly reduced. Conclusion APOBEC3G eukaryotic expression vector pcDNA3.1-A3G was constructed successfully. APOBEC3G can inhibit HBV replication in vitro, which provided the experimental basis for further study of APOBEC3G as an anti-HBV drug.