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目的:构建APOBEC3G真核表达载体,在体外初步探讨APOBEC3G对HBV复制表达的影响。方法:应用RT-PCR法从人PBMC细胞中扩增APOBEC3G基因,分子克隆法构建表达APOBEC3G蛋白的真核表达载体pcDNA3.1-APOBEC3G,并脂质体法转染HepG22.2.15细胞。Western-blot鉴定细胞中APOBEC3G蛋白的表达。ELISA法检测细胞培养上清液中HBsAg和HBeAg水平,实时定量PCR检测HBVDNA和HBV mRNA水平。结果:经酶切鉴定及测序证实APOBEC3G真核表达载体被成功构建,APOBEC3G蛋白可在HepG22.2.15细胞中表达。转染APOBEC3G重组质粒的HepG22.2.15细胞的HBsAg、HBeAg、HBVDNA及HBV mRNA水平均明显低于未转染重组质粒的HepG22.2.15细胞。结论:APOBEC3G明显抑制了HepG22.2.15细胞中HBV的复制与表达,成功构建APOBEC3G真核表达载体,为深入研究APOBEC3G抗HBV的作用机制奠定实验基础。
OBJECTIVE: To construct APOBEC3G eukaryotic expression vector and to investigate the effect of APOBEC3G on HBV replication in vitro. Methods: APOBEC3G gene was amplified by RT-PCR from human PBMCs. The recombinant plasmid pcDNA3.1-APOBEC3G was constructed by molecular cloning. The recombinant plasmid was transfected into HepG22.2.15 cells by lipofectamine 2000. Western blot analysis of APOBEC3G protein expression in cells. The levels of HBsAg and HBeAg in cell culture supernatants were detected by ELISA, and the levels of HBVDNA and HBV mRNA were detected by real-time quantitative PCR. Results: The APOBEC3G eukaryotic expression vector was confirmed by restriction enzyme digestion and sequencing. APOBEC3G protein was expressed in HepG22.2.15 cells. The levels of HBsAg, HBeAg, HBVDNA and HBV mRNA in HepG22.2.15 cells transfected with APOBEC3G recombinant plasmids were significantly lower than those in HepG22.2.15 cells without recombinant plasmids. Conclusion: APOBEC3G significantly inhibits the replication and expression of HBV in HepG22.2.15 cells, and successfully constructed the APOBEC3G eukaryotic expression vector, which lays the foundation for further studies on the anti-HBV mechanism of APOBEC3G.