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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 construct shRNA eukaryotic expression vector psiHBV targeting precRNA region in HepG2 cells and observe its inhibitory effect on HBeAg expression in HepG2 2.15 cells. This study provides experimental evidence for exploring new measures for preventing and treating HBV infection. Methods Aiming at HBV prec / c gene sequence, shRNA expression vector psiHBV1, psiHBV2 and unrelated sequence psiHBVc were constructed. Co-transfection of psiHBV with lentivirus-helper plasmids 293T cells were infected with HepG2 2.15 cells after assembly of lentivirus particles. Pre / c mRNA transcription was detected by RT-PCR. MEIA was used to detect cell supernatants and cell lysates HBeAg expression. Results The double digestion and sequencing of the recombinant plasmids were consistent with the expected results. The transcripts of prec / c mRNA were reduced after the lentivirus particles were infected by HepG2 2.15 cells. Compared with the control group, the expression of HBeAg was also significantly decreased. The expression of HBeAg The inhibitory effect was statistically significant (P <0.01). Conclusion The lentiviral vectors psiHBV1 and siHBV2 targeting HBVprec / c were successfully constructed and lentivirus-mediated RNAi can inhibit the expression of HBV, providing an experimental basis for the application of RNA interference in the treatment of hepatitis B.