摘要
目的针对乙型肝炎病毒(HBV)prec/c区构建shRNA表达载体,观察shRNA表达载体对HBV抗原表达的抑制作用,为运用RNA干扰(RNAi)技术治疗乙型肝炎奠定基础。方法运用基因重组技术构建shRNA表达载体,经酶切,测序鉴定确认后,与1.5倍HBV真核表达质粒pHBV1.5共转染Hela细胞,并于转染后72 h,用微粒子酶免疫实验(MEIA)分别检测细胞上清液和细胞裂解液中HBsAg和HBeAg表达水平;用半定量PCR检测prec/c mRNA的转录情况;分析shRNA表达载体对HBe/HBs抗原表达的抑制情况。结果成功构建了针对HBV prec/c区的shRNA表达载体psiHBV4、psiHBV5、psiHBV6及无关shRNA表达载体psiC;筛选到对HBeAg有明显的抑制作用的psiHBV4载体,同时其对HBsAg的表达有促进作用;psiHBV5和psiHBV6对HBeAg的抑制作用相对较弱,其对HBsAg的表达也有不同程度的促进作用;无关干扰对照psiC对HBV两种抗原的表达均无显著抑制作用。结论成功构建带U6启动子的shRNA表达载体并初步筛选到可显著抑制HBeAg表达的psiHBV4;本研究设计的3个靶向序列中,只有一个序列有大约70%的抑制率,而另外两个序列的抑制率相对较小,说明RNA干扰作用有较强的序列依赖性;无关干扰序列psiC几乎无干扰作用,说明siRNA产生的干扰作用具有高度的特异性。以上结果为应用RNAi治疗乙型肝炎奠定了一定的基础。
Objective To construct the vector expressing shRNA that specifically targets the pre-core gene sequence of hepatitis B virus and investigate its inhibitory effect on the expression of hepatitis B virus antigen. Methods The recombinant vectors that expressing shRNA was constructed through gene recombination and confirmed with restriction enzyme digestion and DNA sequencing. Then, the eukaryotic expression plasmid pHBV1.5 containing 1.5-fold-overlength genome of HBV and the vectors expressing shRNA were co-transfected into Hela cells. At 72 hours post transfection, the levels of HBsAg and HBeAg in the cell culture medium and cell lysate were determined by microparticle enzyme immunoassay （MEIA） with Abbot kits. Prec/c mRNA was analyzed by semi-quantity PCR. Results We successfully constructed the vectors expressing shRNA that could specifically target the pre-core gene sequence of hepatitis B virus and the vector expressing shRNA that had nothing to do with HBV. psiHBV4 could efficiently inhibit the expression of HBeAg, as well as promotion the expression of HBsAg. Whereas the other two vectors expressing shRNA exhibited less inhibitory effect on the replication and the expression of HBeAg but promoted the expression of HBsAg at different extent. The control vector psiC has no effects on HBV. Conclusion The psiHBV4 expressing shRNA that specifically targeted the pre-core gene sequence of hepatitis B virus could exert robust inhibitory effects on HBeAg expression, which suggests that RNAi strategy may represent a potentially efficacious approach to the clinical management of HBV infection.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第3期311-315,共5页
Immunological Journal
基金
四川省教育厅重点课题(2003A066)
