慢病毒介导基于microRNA系统的HBs RNAi技术抑制HBV复制

Lentiviral vector-mediated RNA interference of HBs gene inhibits replication of HBV

目的:构建HBs基因RNAi慢病毒载体,观察其对HBV复制和抗原表达的作用。方法:针对已经筛选确定的HBs基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经MluⅠ和ClaⅠ酶切后的pGCLM-GFP载体连接产生慢病毒载体,PCR筛选阳性克隆,测序鉴定。用pGCLM-GFP、pHelper1.0和pHelper2.0质粒共转染包装细胞293T,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。培养HepG2.2.15细胞系,用慢病毒(MOI=1和MOI=10)对肝癌细胞进行感染,感染后细胞培养上清进行ELISA、Western印迹、HBV DNA定量分析。结果:PCR和测序结果证实,成功构建HBsshRNA的慢病毒载体。包装慢病毒后浓缩病毒悬液的滴度为5×108～2×109TU/ml。慢病毒HBsRNAi后,对HBV复制和抗原表达的抑制作用显著。感染4d后,抑制效应开始出现,一直持续到第9天,抑制效应达到高峰(P<0.05)。相对于阴性对照,HBs shRNA作用后细胞上清中HBsAg分泌量下降70%以上(P<0.05),而Western印迹和real-time PCR结果进一步证实了上述结果,在蛋白水平和mRNA水平都得到了进一步验证。经HBV DNA定量,发现慢病毒RNAi后DNA水平也显著下降(P<0.05)。结论:成功构建HBs基因RNAi慢病毒载体,以HBs基因为靶点的慢病毒介导的基于micro RNA系统的RNAi技术能有效抑制HBV的复制和抗原表达。

Objective：To construct a lentiviral vector（LV） of RNA interference （RNAi） targeting HBs gene and to observe its effect on the replication of HBV and expression of antigens. Methods： The effective sequence of siRNA targeting HBs gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the lentivirus vector （pGCLM-GFP）. The resulting lentivirus vector containing HBs shRNA was named LVshHBs, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentivirus vector pGCLM-GFP, pHelper 1. 0 and pHelper 2. 0. All virus stocks were produced by calcium phosphate- mediated transfection. The titer of virus was tested according to the expression level of GFP. HepG2.2.15 cells were infected with LVshHBs and the supernatant of the cells was subjected to ELISA, Western blotting analysis and HBV DNA quantitative analysis. Results： PCR and DNA sequencing demonstrated that the LVshHBs-produeing HBs shRNA was successfully constructed. The titer of concentrated virus was5×10^8～2×10^9 TU /ml. The inhibitory effect was efficient and the corresponding viral transcript and expression of antigens were decreased after infection. The inhibitory effect was observed 4 days after infection and peaked 9 days after the initial treatment with RNAi. Secreted HBsAg was reduced by 〉70% in cell culture compared with the negative control, which is also confirmed by Western blotting and real-time PCR. After quantification of HBV DNA, the level of DNA relative to the controls was also significantly reduced after RNAi treatment（P（0.05）. Conclusion： The lentivirus RNAi vector of HBs has been successfully constructed. The lentiviral microRNA-based RNAi targeting HBs can specifically mediate the down-regulation of HBs expression, inhibiting HBV replication and antigen expression.