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目的构建针对乙型肝炎病毒(HBV)X基因的siRNA表达载体,观察其对HBV基因表达和复制的影响。方法设计并合成针对HBVX区基因的siRNA寡核苷酸,经退火形成双链后克隆入pSUPER载体,构建成功的siRNA表达载体与pTK-Hyg质粒共转染稳定表达HBV的HepG22·2·15细胞,潮霉素抗性筛选获得稳定细胞克隆,对所得细胞培养上清液中的HBsAg和HBeAg进行定量检测,逆转录-聚合酶链反应(RT-PCR)检测靶基因mRNA的抑制效果,荧光定量PCR检测HBVDNA。结果成功构建了针对HBVX基因的siRNA表达载体pSUPER-X1和pSUPER-X2,两种siRNA均能明显抑制HepG22·2·15细胞的HBsAg和HBeAg分泌,抑制率分别为97%和88%,RT-PCR结果显示HBV的mRNA表达降低,荧光定量PCR结果证实siRNA能降低HBVDNA拷贝数2个数量级。结论载体产生的针对HBVX基因的siRNA能高效、特异地抑制HBV基因的表达和复制。
Objective To construct a siRNA expression vector targeting to hepatitis B virus (HBV) X gene and observe its effect on HBV gene expression and replication. Methods siRNA oligonucleotides targeting HBVX region were designed and synthesized. After double-stranded annealing, they were cloned into pSUPER vector. The constructed siRNA expression vector and pTK-Hyg plasmid were co-transfected into HepG22.2.15 cells stably expressing HBV , And hygromycin resistance screening. The HBsAg and HBeAg in the cell culture supernatant were detected quantitatively, and the inhibitory effect of target gene mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The fluorescence quantitative PCR detection of HBVDNA. Results The siRNA expression vectors pSUPER-X1 and pSUPER-X2 targeting HBVX gene were successfully constructed. Both siRNAs could significantly inhibit the secretion of HBsAg and HBeAg in HepG22.2.15 cells with the inhibition rates of 97% and 88%, respectively. RT- The result of PCR showed that the mRNA expression of HBV was decreased. Fluorescent quantitative PCR confirmed that siRNA could reduce the HBV DNA copy number by two orders of magnitude. Conclusion siRNA targeting HBVX gene can efficiently and specifically inhibit HBV gene expression and replication.