慢病毒介导基于microRNA系统的HBs RNAi技术抑制HBV复制

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HBs shRNA的慢病毒载体。包装慢病毒后浓缩病毒悬液的滴度为5×108~2×109 TU /ml。慢病毒HBs RNAi后,对HBV复制和抗原表达的抑制作用显著。感染4 d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microRNA系统的RNAi技术能有效抑制HBV的复制和抗原表达。

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

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...