Skp2-siRNA重组腺病毒载体构建及鉴定

目的:构建针对Skp2的siRNA腺病毒载体。方法:合成针对Skp2的siRNA靶DNA序列及相应阴性对照序列,退火成DNA双链,然后亚克隆穿梭质粒pShuttle-H1,Pme I线性化后,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染AD293细胞,包装得到pAd-Skp2-siRNA和pAd-Skp2-siRNA-NC重组腺病毒。用病毒体外感染结肠癌SW480细胞,Western blot检测Skp2蛋白表达水平。结果:重组腺病毒载体经酶切、鉴定正确,制备的病毒感染效率高,能显著抑制Skp2蛋白表达。结论:细菌内同源重组成功构建了Ad-Skp2-siRNA重组腺病毒及阴性对照病毒。

Construction and Identification of Recombinant Adenovirus Vector of siRNA Targeting Skp2

Objective To construct an adenovirus vector expressing small interfering RNA(siRNA) targeting to Skp2.Methods The siRNA containing DNA sequence targeting to Skp2 and its negative control sequence were designed,synthesized,annealed and subcloned into pShuttle-H1 containing the green fluorescent protein.The linearized shuttle plasmid by Pme I was recombined with back-bone pAdEasy-1 in BJ5183 bacteria.The recombinant pAd-Skp2-siRNA adenovirus particles were produced by transfection of AD293 cells and subsequently infected colonic cancer SW480 cells.The Skp2 protein levels were detected by Western blot.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing,the infection efficiency of constructed adenovirus was high,and could obviously inhibit the expression of Skp2.Conclusion The recombinant pAd-Skp2-siRNA and negative control bacterial were successfully constructed by bacterial homologous recombination.