抑制RAGE表达的siRNA重组腺病毒载体的构建及鉴定

Recombinant Adenovirus Vector Construction of siRNA Targeting RAGE and its Functional Characterization

目的构建特异性针对细胞膜上晚期糖基化终末产物受体（RAGE）的小干扰RNA（siRNA）腺病毒载体，鉴定其对大鼠胰岛β细胞系INS-1细胞RAGE表达的影响。方法设计并合成针对大鼠RAGE基因的siRNA的靶DNA序列，克隆于穿梭载体pAdTrack中，与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组，脂质体法转染至QB1293A细胞中包装，获得RAGE—siRNA的重组腺病毒，荧光显微镜观察RAGE—siRNA感染INS-1细胞后绿色荧光蛋白（GFP）的表达，Western印迹检测RAGE的蛋白表达。结果成功制备RAGE—siRNA重组腺病毒，在INS-1细胞中RAGE—siRNA感染效率达到90％以上，并能够抑制INS-1细胞RAGE的表达。结论成功构建了携带RAGE的siRNA重组腺病毒载体，能有效沉默INS-1细胞中RAGE的表达。

Objective To construct an adenovirus vector expressing small interfering RNA （siRNA） against rat RAGE （Receptor of Advanced glycation end products） which silences RAGE gene expression in Pancreatic β-cell line INS-1 cells. Methods The RAGE template DNA sequences were designed by online tools. Then the sense and antisense siRNA oligonucleotide templates were chemically synthesized, annealed and cloned into adenoviral shuttle vector pAdTrack. The recombinant adenovirus vector pAd-RAGE-siRNA was obtained by homologous recombination with pAdTraek and pAdeasy-1 in bacteria strain BJ5183. The reeombined adenovirus was produced in QBI293A cells and subsequently transduced rat pancreatic beta-cell line INS-1 cells. Green Fluorescent protein （GFP） was observed to monitor the infection efficiency using inverted Fluorescent Microscope. The RAGE protein levels in INS-1 cells were detected by western blot. Results The adenovirus vector expressing small intefering RNA for RAGE gene could specifically shutdown the endogenous RAGE protein expression in INS-1 cells. Conclusion The recombined adenovirus RAGE-siRNA can effectively downregulate RAGE expression in INS-1 cells.