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目的构建针对乙型肝炎病毒(HBV)S基因的siRNA(shortinterferingRNA)表达载体pSUPER-S1和pSUPER-S2,观察其对HepG22.2.15细胞中的HBV基因表达的影响。方法设计并合成针对HBVS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,经200μg/ml潮霉素抗性筛选,4周后获得稳定细胞克隆,对所得细胞培养上清中的HBsAg和HBeAg进行定量检测,免疫荧光染色检测细胞内抗原的表达,同时用RT-PCR检测靶基因mRNA的抑制效果。结果成功构建了针对HBVS基因的siRNA表达载体pSUPER-S1和pSUPER-S2,两种siRNA均能明显抑制HepG22.2.15细胞的HBsAg和HBeAg分泌,抑制率分别为83%和78%,免疫荧光染色显示siRNA能抑制细胞内抗原的合成,RT-PCR结果证实HBV的mRNA表达降低,而无关序列的siRNA和对照则无此作用。结论载体产生的针对HBVS基因的siRNA能稳定、高效、特异地抑制HBV基因的表达。
Objective To construct the shortinterfering RNA expression vectors pSUPER-S1 and pSUPER-S2 against Hepatitis B virus (HBV) S gene and study the effect of HBV on HBV gene expression in HepG22.2.15 cells. Methods siRNA oligonucleotides targeting HBVS gene were designed and synthesized. After annealing, the double-stranded DNA was cloned into pSUPER vector. The constructed siRNA expression vector and pTK-Hyg plasmid were co-transfected into HepG22.2.15 cells stably expressing HBV. 200μg / ml hygromycin resistance screening, stable cell clones were obtained after 4 weeks, the detection of HBsAg and HBeAg in the resulting cell culture supernatant quantitative detection of immunofluorescence staining of intracellular antigen expression, and RT-PCR detection target Inhibition of gene mRNA. Results The siRNA expression vectors pSUPER-S1 and pSUPER-S2 targeting HBVS 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 83% and 78%, respectively. Immunofluorescence staining siRNA can inhibit the synthesis of intracellular antigens, RT-PCR results confirmed that HBV mRNA expression decreased, while unrelated sequence siRNA and control did not. Conclusion siRNA targeting HBVS gene can inhibit HBV gene expression stably, efficiently and specifically.