HBV prec/c shRNA慢病毒真核表达载体的构建和鉴定

Construction and appreciation of eukaryotic expression vector of HBV prec/c shRNA

目的构建针对HBV prec/c区的shRNA真核表达载体psiHBV,感染HepG2 2.15细胞后观察其对HBeAg表达的抑制作用,为探讨防治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与慢病毒辅助系统质粒共转染293T细胞组装慢病毒颗粒后,感染HepG2 2.15细胞,RT-PCR检测prec/c mRNA的转录,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。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)。结论成功构建针对HBVprec/c的慢病毒载体psiHBV1、siHBV2,慢病毒介导的RNA i能抑制HBV表达,为应用RNA干扰技术治疗乙型肝炎提供了实验依据。

Objective To investigate new measures to prevent and cure the infection of hepatitis B virus and construct eukaryotic expression vectors shRNA aiming directly at the pre-core gene sequence of hepatitis B virus through observing its effect on HBeAg expression in HepG22.15 cells.Method The shRNA express vectors of psiHBV1,psiHBV2 and psiHBVc aiming directly at the pre-core gene sequence of hepatitis B virus were constructed.After psiHBV with lentiviral vector auxilary system was transfected into 293T cells,the lentiviral 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 RT-PCR and MEIA respectively.Result The identification of shRNA by enzyme cutting and sequencing was coincided with the anticipation.After the lentiviral particles were assembled and HepG2 2.15 cells infected the transcription of prec/c mRNA and the expression of HBeAg decreased greatly.Compared with control group the inhibition of lentiviral particles to expression of HBeAg showed significant deviation（P0.01） Conclusion The lentiviral vectors psiHBV1,psiHBV2 and psiHBVc were constructed succesfully.RNAi mediated by lentivirus can control the expression of HBV.It provides a basis for the application of RNA interference in treatment of hepatitis B.