小环载体介导的siRNA 稳定抑制乙肝病毒的复制和表达

A minicircle DNA vector-mediated siRNA to stably suppress hepatitis B virus replication and expression

摘要:

摘要:【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。

Abstract:

Abstract:［Objective］We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV ) replication and gene expression in vitro． ［Methods］ siRNA targeting HBV Sgene (siHBS) was designed ，synthesized and cloned into a minicircle DNA vector pMC． BESPX-MCS2. After sequencing，we transformed the recombinant pMC-H1-siHBS-U6 into E.coli ZYCY10P3S2T， and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium． As expected，a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette． Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1. 3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells． ［Results］We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6，which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks． Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%． ［Conclusion］ The minicircle DNAbased RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically，efficiently and steadily． Thus，this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection．