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。
[ 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 S gene （siHBS） was designed , synthesized and cloned into a minicircle DNA vector pMC. BESPX-MCS2. After sequencing, we transformed the recombinant pMC-HI-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-HI-siHBS-U6 was generated only consisting of gene expression cassette. Then pmc-HI-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 minicirele-based RNAi vector pmc-HI-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 DNA- based RNAi vector pmc-HI-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.
出处
《微生物学报》
CAS
CSCD
北大核心
2012年第2期191-197,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金(81071770)~~
