靶向X区的siRNA抑制乙型肝炎病毒基因的表达和复制  

Inhibition of hepatitis B virus expression and replication by vector-based siRNAs targeting X gene

作　　者：刘家云 郝晓柯 李庆霞[2] 黄红艳[2] 马龙洋[2] 温伟红[3] 贾林涛[2] 杨安钢[3] 

机构地区：[1]第四军医大学第一附属医院检验科,西安710032 [2]第四军医大学生物化学与分子生物学教研室 [3]第四军医大学免疫学教研室

出　　处：《中华检验医学杂志》2006年第12期1084-1088,共5页Chinese Journal of Laboratory Medicine

基　　金：国家973重点基础研究发展规划基金项目(2004CB518805);国家863高技术研究发展计划基金项目(2004AA217071);国家自然科学基金资助项目(30500433)

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 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.

关 键 词：肝炎病毒 乙型 RNA 小分子干扰 转染 聚合酶链反应 

分 类 号：R346[医药卫生—基础医学]