人miR-155真核过表达载体的构建及其对HepG2.2.15细胞中HBeAg的抑制效应

Construction of a eukaryotic vector expressing human miR-155 and inhibitory effect of miR-155 on HBeAg in HepG2.2.15 cells

目的:构建人microRNA-155(miR-155)真核过表达载体,并探讨其对HepG2.2.15细胞中乙型肝炎e抗原(hepatitis B e antigen,HBeAg)的抑制作用,为研究其基因调控机制对乙型肝炎病毒(hepatitis B virus,HBV)复制及免疫状态影响提供实验基础.方法:以人肝癌细胞系HepG2.2.15细胞基因组为模板,通过PCR扩增人miR-155前体序列,酶切后连接到pmR-mCherry质粒,构建pmiR-155真核过表达载体,然后进行酶切和测序鉴定.利用脂质体将pmiR-155转染HepG2.2.15细胞,同时设空质粒(pmR-mCherry质粒)转染组和空白对照组.转染24 h后荧光显微镜下观察细胞内Cherry表达,RT-PCR检测各组细胞内miR-155表达量以及ELISA法检测HBeAg分泌量的改变.结果:经测序证实,成功构建人pmiR-155真核过表达载体.转染细胞后进行应荧光观察,载体中Cherry有较好的表达活性.RT-PCR表明,与对照组细胞相比,pmiR-155组细胞内所表达的miR-155明显提高.ELISA结果示pmiR-155组细胞所分泌的HBeAg量显著低于空载组和空白组.结论:成功构建了人miR-155真核过表达载体;转染HepG2.2.15细胞后表达稳定;蛋白水平检测表明其可以抑制HepG2.2.15细胞中HBeAg的表达.

AIM： To construct an eukaryotic vector carrying human microRNA-155（miR-155） and to analyze the inhibitory effect of miR-155 on HBeAg in HepG2.2.15 cells.METHODS： The pre-miR-155 was amplifiedfrom total DNA of human hepatoma cell line HepG2.2.15 by PCR. The target gene fragment was digested with EcoRⅠ and BamHⅠ, and cloned into the pmR-mCherry plasmid. Restriction diges-tion and DNA sequencing were performed to eval-uate the recombinant vector. miR-155 was trans-fected into HepG2.2.15 cells by liposome-mediated method. The cells transfected with empty plasmid and untransfected cells were used as controls. The expression of cherry was detected by fluorescence microscopy after 24 h. The intracellular expression of miR-155 was detected by RT-PCR. ELISA was carried out to analyze the levels of HBeAg.RESULTS： The pmiR-155 eukaryotic expression vector was successfully constructed. Fluorescence microscopy showed that the cherry protein was expressed in the HepG2.2.15 cells. miR-155 level in HepG2.2.15 cells transfected with the recombi-nant plasmid was significantly higher than those in controls. Compared with cells transfected with empty plasmid and untransfected cells, specific miR-155 could significantly decrease HBeAg gene expression in HepG2.2.15 cells.CONCLUSION： A recombinant plasmid express-ing miR-155 has been successfully constructed, and miR-155 is expressed stably in HepG2.2.15 cells. miR-155 can inhibit the expression of HBeAg in HepG2.2.15 cells.