人γ-干扰素真核表达载体的构建及其体外抑制HBV的作用

Construction of a eukaryotic expression vector expressing human IFN-γ and its inhibitory effect on HBV replication in vitro

目的观察携带人γ干扰素(IFN-γ)基因的真核表达载体(pcDNA3.1-IFN-γ)体外转染hepG2.2.15细胞后抑制乙型肝炎病毒(HBV)的作用。方法利用PCR技术和基因重组方法构建表达人IFN-γ的真核表达载体,体外转染hepG2.2.15细胞后收集细胞培养上清液,用ELISA法检测人IFN-γ蛋白的分泌量,并分别用荧光定量PCR和ELISA试剂盒检测细胞培养上清中病毒释放的HBV-DNA和HBeAg、HBsAg抗原含量。结果转染pcDNA3.1-IFN-γ组细胞HBeAg含量比空载体阴性对照组和空白2.2.15细胞对照组下降了49%,而两个对照组间HBeAg含量无显著差异;HBsAg含量比空载体阴性对照组下降了35%、比空白2.2.15细胞对照组下降了33%,两个对照组间HBsAg含量无显著差异。同时,转染pcDNA3.1-IFN-γ组细胞上清中HBVDNA含量比两个对照组均显著降低,而对照组间HBVDNA含量无显著差异。结论成功构建了人IFN-γ真核表达载体并可以在体外有效抑制HBV复制,为将人IFN-γ应用于抗HBV的基因治疗奠定基础。

Objective To observe the inhibitory effect of a eukaryotic expression vector expressing human IFN-γ （pcDNA3.1- IFN-γ） on HBV replication in hepG2.2.15 cells. Methods The eukaryotic expression vector expressing human IFN-γ was constructed using PCR and gene recombination technique. hepG2.2.15 cells were transfected with pcDNA3.1-IFN-γ and the culture supernatant was collected to determine the expression of IFN-γ protein by ELISA. The HBV DNA copies and the concentration of HBeAg and HBsAg were measured by fluorescence real-time PCR and ELISA kit, respectively. Results Compared with that of negative control and blank 2.2.15 cells, the concentration of HBeAg in the supernatant of 2.2.15 cells transfected with pcDNA3.1- IFN-γ were decreased by 49%, and HBsAg concentration was lowered by 35% and 33%, respectively. A significant decrease of HBV DNA copies was observed in pcDNA3.1- IFN-γ-transfected cells in comparison with the two control cells. No significant differences were noted in all the results between the two control groups. Conclusion We have successfully constructed the eukaryotic expression vector expressing human IFN-γ, which provides a basis for anti-HBV gene therapy using human IFN-γ