P27特异性siRNA重组腺病毒载体的构建及功能鉴定

目的构建针对P27的siRNA腺病毒载体及相应的对照病毒载体，并鉴定重组腺病毒在小鼠胰岛中对内源性皿7基因表达的影响。方法合成针对p27的siRNA的靶DNA序列及相应的阴性对照序列，连接至pShuttle-H1质粒中，然后用NotI和HindHI限制性内切酶将H1-siRNA片段从pShuttle—H1-siRNA质粒上双酶切下来，克隆至空载pAdTrack穿梭质粒上，与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组，转染QBI-293A细胞，包装得到pAd—P27-siRNA和pAd—NC重组腺病毒。用病毒体外感染小鼠胰岛，Western印迹法检测P27蛋白表达水平。结果重组腺病毒载体经测序鉴定正确，制备的病毒感染效率高，能有效抑制小鼠胰岛中P27的表达。结论成功构建了针对P27的siRNA重组腺病毒载体，为进一步研究P27在胰岛β细胞生长中的作用提供了基础。

Recombinant Adenovirus Vector Construction of siRNA Targeting P27 and its Functional Characterization

Objective To construct an adenovirus vector that expresses small interfering RNA （siRNA） against P27 to shutdown its gene expression. Methods The sense and antisense siRNA oligonucleotide templates were chemically synthesized, annealed and subcloned into pShuttle-H1. To allow infection efficiency to be conveniently monitored, the HI-siRNA fragments were cut from pShuttle-H1 and ligated into the pAdTrack plasmid upstream of the CMV-green fluorescent protein （GFP） cassette. The pAdTrack-Hl-siRNA plasmid was recombined with back-bone pAdEasy-1 in BJ5183 bacteria. The recombinant pAd-P27-siRNA adenovirus particles were produced by transfection of QBI-293A cells and subsequently infected mouse islets. The P27 protein levels were detected by Western blot. Results The adenovirus vector expressing small interfering RNA for P27 gene could specifically shutdown the endogenous P27 protein expression in mouse islets. Conclusion The recombinant pAd-P27-siRNA was successfully constructed. P27; siRNA; adenoviral vector