腺相关病毒载体介导siRNA抑制HBV的表达与复制

Inhibition of Hepatitis B Virus by Small Interfering RNA Expressed from Adenovirus-associated Virus Vectors

目的:构建并制备能够有效抑制HBV mRNA表达的siRNA-重组腺相关病毒。方法:将筛选到的siRNA片段克隆入骨架质粒pAAV-MCS中,与pAAV-RC和pHelper质粒共转染人胚肾293T细胞,包装出重组腺相关病毒,将纯化后的重组病毒直接感染HepG2.2.15细胞,通过酶联免疫法和荧光定量PCR法检测重组腺相关病毒的抑制效果。结果:重组腺相关病毒显著降低HBV分泌的相关抗原蛋白以及DNA和RNA水平。结论:重组腺相关病毒载体介导的siRNA能够有效抑制HBV的表达与复制。

Objective：To construct the recombinant Adenovirus-associated virus（AAV） vector for siRNA targeting HBV mRNA and to evaluate the inhibitive effect on Hep G 2.2.15 cell.Method：The packing cell line（human embryonic kidney 293T cell） was co-transfected with AAV backbone plasmid pAAV-MCS which has been cloned into the selected siRNA,along with the pAAV-RC and pHelper in AAV Helper-Free System.The recombinant adenovirus-associated virus was packaged and amplified,followed by infection of the Hep G 2.2.15 cells.The expression level of HBV gene and the replication of HBV were analyzed by ELISA and fluorescence quantitative RCR.Result：The recombinant adenovirus-associated virus vectors containing siRNA targeting HBV mRNA was successfully constructed.ELISA results demonstrated that the siRNA can effectively inhibit the secretion of HBsAg and HBeAg and the fluorescence quantitative RCR results also showed that the copies of HBV DNA and RNA were significantly reduced.Conclusion：The recombinant adenovirus-associated virus vector can effectively inhibit the expression and replication of HBV in vitro.