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RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默现象, 是近年来兴起的一项研究生物基因调控与功能的崭新技术. 庚型肝炎病毒(HGV)是一单正链RNA病毒, 复制时不与宿主细胞基因组整合, 尤其适合用于RNAi的研究. 构建了含HGV完整结构基因并携带筛选标志潮霉素基因的真核表达载体pVAX.EH, 转染Huh-7细胞后, 筛选获得稳定表达HGV 结构蛋白的Huh-7细胞株(Huh-7-EH). RT-PCR和Western blot检测证实, HGV结构基因能在Huh-7-EH细胞中转录、表达, 并能进行剪切和翻译后修饰. 以体外转录法制备了2对靶向HGV E2基因的siRNA(1-E2 siRNA和2-E2 siRNA), 将其导入Huh-7-EH细胞中, 采用Western blot和克隆形成实验证实, HGV 1-E2 siRNA和2-E2 siRNA均能特异性抑制HGV结 构蛋白的表达, 抑制作用可维持1周以上. 其中2-E2 siRNA的抑制作用更强, 转染后对Huh-7-EH细胞潮霉素抗性克隆形成的抑制率达到了99%. Huh-7-EH细胞转染siRNA后对潮霉素敏感, 说 明HGV E2 siRNA不仅使HGV E2区的mRNA降解, 还可使融合在HGV E2区下游的潮霉素mRNA降解. 综上所述, 本实验建立的稳定表达HGV结构蛋白的Huh-7-EH细胞株, 能作为用于研究HGV复制和RNAi的细胞模型; HGV结构基因区的siRNA可同时抑制HGV结构蛋白及其?
RNA interference (RNAi) is a novel technology to study the regulation and function of biological genes in recent years due to the post-transcriptional gene silencing of homologous genes triggered by small interfering RNAs (siRNAs) HGV) is a single positive-stranded RNA virus that is not integrated with the host cell genome during replication and is especially suitable for RNAi research.We constructed an eukaryotic expression vector pVAX.EH containing the complete structural gene of HGV and carrying the selection marker hygromycin gene Huh-7 cells (Huh-7-EH) stably expressing HGV structural protein were screened and transfected into Huh-7 cells.The results of RT-PCR and Western blot confirmed that the HGV structural gene could be expressed in Huh-7-EH cells (1-E2 siRNA and 2-E2 siRNA) targeting HGV E2 gene were prepared by in vitro transcription and introduced into Huh-7-EH The results of Western blot and clonogenic assay confirmed that both HGV 1-E2 siRNA and 2-E2 siRNA could specifically inhibit the expression of HGV structural protein, and the inhibitory effect could be maintained for more than 1 week. The inhibitory effect of 2-E2 siRNA was more Strong, suppression of Huh-7-EH cell hygromycin-resistant clones after transfection Rate reached 99%. Huh-7-EH cells were sensitive to hygromycin after transfected with siRNA, indicating that HGV E2 siRNA not only degrades the mRNA of HGV E2 region, but also degrades the hygromycin mRNA fused downstream of HGV E2 region In conclusion, the Huh-7-EH cell line stably expressing the HGV structural protein established in this study can be used as a cell model for studying HGV replication and RNAi. The siRNA for HGV structural gene region can simultaneously inhibit HGV structural protein and its?