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目的以乙型肝炎病毒(HBV)S基因区为靶位,构建表达siRNA的质粒载体pSilencer3·1-H1hygro,体外观察siRNA抗HBV的效果。方法以HepG2·2·15细胞为靶细胞,利用脂质体Metafectene转染表达siRNA的质粒载体pSilencer3·1-H1hygro于细胞中,用时间分辨免疫荧光分析法(IFMA法)检测细胞上清中HBsAg和HBeAg,用定量聚合酶链反应(FQ-PCR)检测细胞上清DNA,用逆转录(RT)-PCR检测HBVmRNA。结果成功构建了表达siRNA的转录质粒载体,siRNA可抑制HBV的抗原表达和病毒复制,1、2、4μgsiRNA对HBsAg的抑制率分别为75%、82%、89%;对HBeAg的抑制率分别为32%、38%、43%;对HBVDNA的抑制率分别为30%、43%、49%;对HBVRNA的抑制率分别为30%、70%、90%。结论靶向HBVS区的siRNA能抑制HBV的抗原表达和复制;siRNA抑制作用呈剂量依赖性和序列特异性。
Objective To construct the plasmid vector pSilencer3.1-H1hygro which targets siRNA for hepatitis B virus (HBV) S gene region and to observe the anti-HBV effect of siRNA in vitro. Methods HepG2-2.15 cells were used as target cells. The plasmid pSilencer3.1-H1hygro was transfected into the cells by lipofectamine Metafectene, and the expression of HBsAg in the supernatant of the cells was detected by IFMA (time-resolved immunofluorescence assay) And HBeAg, the cell supernatant DNA was detected by quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA was detected by reverse transcription (RT) -PCR. Results The siRNA plasmid vector was successfully constructed. SiRNA inhibited HBV antigen expression and viral replication. The inhibitory rates of 1,2,4μgsiRNA on HBsAg were 75%, 82% and 89%, respectively. The inhibitory rates of HBeAg were 32%, 38% and 43%, respectively. The inhibitory rates of HBVDNA were 30%, 43% and 49%, respectively. The inhibitory rates of HBV RNA were 30%, 70% and 90%, respectively. Conclusion siRNA targeted at HBVS can inhibit the expression and replication of HBV antigen in a dose-dependent and sequence-specific manner.