rAAV载体介导的HDV核酶对HBV基因表达的抑制作用

目的探讨重组腺相关病毒介导的HDV核酶在细胞内抑制乙型肝炎病毒基因表达的作用。方法将针对HBV基因序列C区的反式HDVR z与U6启动子和绿色荧光蛋白基因EGFP及其启动子CMV共同插入腺相关病毒载体pSNAV中并取代其原有CMV启动子区域,构建为pSNAV-CR z重组载体。并将pSNAV-CR z、pSNAV及pLEGFP质粒转染HepG2.2.15培养细胞,荧光显微镜下观察绿色荧光,对转染后培养细胞内蛋白进行HBeAg和HBsAg的ELISA分析。结果所构建的重组载体经双酶切及PCR验证与实验设计一致。重组载体转染培养细胞后,荧光显微计数表明,pSNAV-CR z的转染效率为30%。HBeAg和HBsAg的ELISA检测显示,pSNAV-CR z重组载体对HepG2.2.15细胞中HBV病毒的HBeAg和HBsAg表达抑制率分别为63.5%和50.07%。结论细胞水平试验证明,重组腺相关病毒载体介导的反式HDVR z在体外培养细胞水平对HBV的复制起到一定的抑制作用。

The inhibition of HBV gene expression HepG cell by rAAV mediated HDV ribozyme

Objective To investigate the HBV gene expression inhibited by adenoassociated virus vector recombinated with HDV ribozyme.Methods pSNAV-CRz recombinant vector was constructed by inserting HDV ribozyme,U6 promoter and EGFP into pSNAV backbone,and then transfected into HepG2.2.15 cells.Transfection efficiency and expressions of S and E antigens of HBV were detected by ELISA analysis.Results The construction of the recombinant vector was identified by restriction enzyme double digestion and PCR.Transfection efficiency of pSNAV-CRz plasmid was 30% according to the cell count under fluorescence microscope.The ELISA test showed that the inhibition ratios of HBeAg and HBsAg expressions were 63.5% and 50.07%,respectively.Conclusion The ELISA result demonstrates that the recombinant adeno-associated virus vector plays an inhibitory role in cell level.