Construction of recombinant adenovirus expressing the short hairpin RNA of rat angiotensin IIreceptor subtype 1

Objective To construct the adenovirus vector expressing the shRNA of rat angiotensin Ⅱ receptor and amplify the adenovirus vector in 293 cells.Methods The rat AT1-shRNA segments was obtained from plasmid pGenesil-1-AT1-shRNA which was constructed at an earlier date by RT-PCR and then was inserted into linearized PUC18 plasmid.After having been screened,the constructed PUC18-AT1-shRNA plasmid was digested with restriction endonucleases and cloned into the shuttle plasmid pDC316 to form the pDC316-AT1-shRNA vector.The pDC316-AT1-shRNA plasmid was cotransfected with genomic plasmid pBHGloxΔ1,3Cre into 293 cells to package the recombinant adenovirus.The recombinant adenovirus was transfected into rat glioma C6 cells,and the green fluorescence protein expression was detected.Results Recombinant adenoviral vector Ad-AT1-shRNA was constructed successfully,which was confirmed by restriction enzyme digestion,PCR and GFP expression.Conclusion The recombinant adenoviral vector carrying AT1shRNA is successfully constructed,and will lay the foundation of application of gene therapy to antihypertension.