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.