抑制FoxO1基因表达的siRNA重组腺病毒载体的构建及功能鉴定

Recombinant adenovirus vector construction of siRNA targeting FoxO1 and its functional characterization

目的构建针对转录因子FoxO1的siRNA腺病毒载体,并鉴定重组腺病毒在人肝癌细胞系HepG2中对内源性FoxO1基因表达的影响。方法设计并合成针对FoxO1的siRNA的靶DNA序列,克隆于穿梭载体pAdTrack-CMV中,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染293细胞,包装得到含si-FoxO1的重组腺病毒,体外转染人肝癌细胞系HepG2,Westernblot检测FoxO1蛋白表达水平的变化。结果成功构建针对FoxO1的siRNA重组腺病毒载体,该重组腺病毒能显著抑制HepG2细胞中FoxO1蛋白的表达(P<0.01)。结论成功构建了针对FoxO1的siRNA重组腺病毒载体,能有效抑制HepG2细胞中FoxO1的表达。

Objective To construct an adenovirus vector that expresses small interfering RNA （siRNA） against FoxO1 to shutdown its gene expression. Methods The FoxO1 template DNA sequence was designed by using online tools. Then the sense and antisense siRNA oligonucleotide templates were chemically synthesized, annealed and cloned into adenoviral shuttle vector pAdTrack-CMV. The recombinant adenovirus vector pAd-si- FoxO1 was obtained by homologous recombination with pAdTrack-CMV and pAdeasy-1 in bacteria BJ5183. The recombined adenovirus was produced in 293 cells and subsequently infected HepG2 cells. The FoxO1 protein levels were detected by Western blot. Results The adenovirus vector expressing small interfering RNA for FoxO1 gene could specifically shutdown the endogenous FoxO1 protein in HepG2 cells. Conclusion The pAd- si-FoxO1 can effectively downregulate FoxO1 expression in HepG2 cell line.