论文部分内容阅读
【目的】尝试构建表达小干扰RNA(small interfering RNA,siRNA)的小环载体,并初步鉴定其对乙肝病毒(hepatitis B virus,HBV)复制及其基因表达的抑制作用。【方法】设计并合成靶向HBV S区的siRNA,将其克隆到小环载体pMC.BESPX-MCS2上,测序正确后将重组体pMC-H1-siHBS-U6转化入感受态E.coliZYCY10P3S2T,然后在培养基中加入L-阿拉伯糖,诱导其降解细菌骨架,获取只含有目的基因表达盒的小环RNA干扰载体pmc-H1-siHBS-U6。将小环RNA干扰载体与HBV真核表达质粒pHBV1.3共转染Huh-7细胞,分别在转染后1-7天,ELISA法检测Huh-7细胞上清中的HBsAg、HBeAg,并且通过Real-time RT-PCR法分析干扰RNA对HBV DNA及mRNA的抑制效果。【结果】成功构建了靶向HBV S基因的siRNA小环表达载体pmc-H1-siHBS-U6。该载体能显著抑制Huh-7细胞HBsAg和HBeAg分泌,并且其抑制效果能够维持2-3周时间。Real-time PCR证实HBV的DNA与mRNA水平分别降低了71%和80%,而对照siRNA及空载体则无此作用。【结论】成功构建了靶向HBV的小环RNA干扰载体,并且其能稳定、高效、特异地抑制HBV基因的表达与复制,该研究不仅对探索HBV的基因治疗提供了重要线索,而且为RNA干扰的应用提供了新的运载体系。
【Objective】 The purpose of this study was to construct a minicircle vector expressing small interfering RNA (siRNA) and preliminary identify its inhibitory effect on hepatitis B virus (HBV) replication and its gene expression. 【Method】 siRNA targeting HBV S region was designed and synthesized. The siRNA was cloned into the minicircle vector pMC.BESPX-MCS2. After sequencing, the recombinant pMC-H1-siHBS-U6 was transformed into competent E. coli ZYCY10P3S2T L-arabinose was added into the medium to induce its degradation of bacterial skeleton, and the small interfering RNA vector pmc-H1-siHBS-U6 containing only the target gene expression cassette was obtained. Huh-7 cells were cotransfected with small loop RNA interference vector and HBV eukaryotic expression plasmid pHBV1.3. The HBsAg and HBeAg in the supernatant of Huh-7 cells were detected by ELISA after 1-7 days of transfection, Real-time RT-PCR analysis of interfering RNA on HBV DNA and mRNA inhibitory effect. 【Result】 The shRNA expression vector pmc-H1-siHBS-U6 targeting HBV S gene was successfully constructed. The vector can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells, and its inhibitory effect can be maintained for 2-3 weeks. Real-time PCR confirmed that HBV DNA and mRNA levels were reduced by 71% and 80%, respectively, whereas control siRNA and empty vector had no effect. 【Conclusion】 The shRNA targeting small interfering RNA (HBV) targeting HBV has been successfully constructed and its expression can be stably, efficiently and specifically inhibited. This study not only provides important clues for exploring the gene therapy of HBV, Interference applications provide a new delivery system.