慢病毒介导的RNA干扰体外抑制HBeAg表达的实验研究  

In vitro investigation of HBeAg expression in human hepatocellular carcinoma cells by lentivirus mediated RNA interference

作　　者：王朝莉[1] 李丽[1] 陈果[1] 唐恩洁[1] 杨致邦[2] 杨尚君[1] 

机构地区：[1]川北医学院分子生物学研究所,四川南充637007 [2]重庆医科大学病原生物学教研室,重庆400016

出　　处：《西部医学》2012年第12期2268-2270,2273,共4页Medical Journal of West China

摘　　要：目的为防治HBV感染的新措施提供实验依据。方法针对HBV prec/c基因序列,构建shRNA表达载体psiHBV1、psiHBV2和无关序列psiHBVc。psiHBV与慢病毒辅助质粒系统共转染293T细胞组装慢病毒颗粒后,感染HepG2 2.15细胞,微粒子化学发光分析仪(MEIA)检测细胞上清和细胞裂解液中HBeAg表达;用定量PCR检测prec/c mRNA的转录情况。结果重组质粒双酶切和测序鉴定与预期结果相符合;组装慢病毒颗粒感染HepG2 2.15细胞后,prec/c mRNA转录降低;与对照组比较,HBeAg的表达水平也显著降低,病毒颗粒对HBeAg表达的抑制作用差异有统计学意义(P<0.01)。结论 RNAi能特异性抑制HBV表达,为应用RNA干扰技术进行乙型肝炎的治疗奠定了基础。Objective To investigate the new measure to prevent and cure from the infection of hepatitis B virus by the lentivirus mediated RNA interference, which aimed directly at the pre core gene sequence of hepatitis B virus. Methods The shRNA express vector of psiHBV1, psiHBV2 and psiHBVc to aim directly at the pre core gene sequence of hepatitis B virus were constructed. After psiHBV with lentiviral vector auxilary system transfected into 293T cells, the lentivira particles were made up and infected HepG22.15 cells. The transcription of prec/c mRNA and the expression of HBeAg in cell supernatant and cell lysate were detected by the methods of Real Time PCR and MEIA respectively. Results The results of the restriction enzyme digestion and sequencing confirmed the vector was constructed successfully. Trausfecting it into HepG2 2.15cells and expressing HBVprec/c mRNA will be controlled in some way. Conclusion RNAi mediated by lentivirus can control the expression of HBV. It provides the experiment similar to RNA interference to treat hepatitis B.

关 键 词：乙型肝炎病毒 RNAI HBEAG 慢病毒 

分 类 号：R512.62[医药卫生—内科学]