APOBEC3G真核表达载体的构建及其对HBV复制表达的影响

Construction of Eukaryotic Expressional Vectors of APOBEC3G and the Effects on the Replication and Expression of HBV

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 eukaryotic expression vector containing APOBEC3G, and to study the effects of APOBEC3G on replication and expression of HBV in vitro. Methods： The APOBEC3G gene was extracted from PBMCs by RT-PCR. Eukaryotic expression vector pcDNA3. 1 APOBEC3G was constructed by molecular cloning and transfected into HepG2 2. 2.15 cells by lipofectamine. The expression of protein APOBEC3G was detected by Western-blot in HepG2 2.2. 15 cells. The levels of HBsAg and HBeAg in the media of transfected HepG22.2. 15 cells were detected by EIASA. The levels of HBV DNA and HBV mRNA were determined by real time PCR. Results： APOBEC3G eukaryotic expression vector was constructed successfully by sequence identification, which could express APOBEC3G protein efficiently in HepG2 2. 2.15 cells. The levels of HBsAg, HBeAg, HBV DNA and HBV mRNA in HepG2 2. 2.15 cells transfected APO- BEC3G recombinant plasmid were significantly lower than that of HepG22. 2.15 cells which were not transfected APOBEC3G. Conclusion： APOBEC3G could obviously inhibit replication and expression of HBV in vitro. The successful construction of APOBEC3G eukaryotic expression vector lays the foundation for further researching the mechanism of anti-HBV effects by APOBEC3G.