中文摘要：

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人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。

英文摘要：

Objective To construct pSUPER vectors expressing short interfering RNAs （siRNA） targeting HBV X gene and to evaluate inhibitory effect of these siRNAs on HBV gene expression and replication in HepG2 2.2. 15 cells. Methods Based on the sequence of HBV in HepG2 2. 2. 15 cells, double sequences targeting HBV X gene were cloned into pSUPER vector. The recombinant plasmids were cotransfected and monoclonal cell strains were screened in hygromycin media. Next, the HBsAg and HBeAg in supematant of the screened cell strains were assayed with Abbott MEIA Kits, HBV DNA was determined by fluorescent quantitative PCR （FQ-PCR）, and HBV mRNA were detected by RT-PCR. Results The pSUPER-X1 and pSUPER-X2 siRNA expression vectors were successfully constructed. The siRNAs could effectively inhibit the secretion of surface antigen and e antigen of HBV in HepG2 2. 2. 15 cells by 97% and 88% respectively, FQ-PCR results revealed that copy of HBV DNA in supematant was significantly reduced and RT-PCR results showed that the HBV mRNA was markedly decreased. Whereas the irrelevant siRNA expression vector and control did not show any inhibitory effect on the expression and replication of HBV. Conclusion These results demonstrate that vector-based siRNAs targeting HBV X gene can stably inhibit HBV replication with great potency and specificity in vitro.