Construction and identification of recombinant adenovirus expressing short hairpin RNA of rat inducible nitric oxide synthase

Abstract

Objective To construct an adenovirus vector expressing short hairpin RNA （shRNA） of rat inducible oxide synthase （iNOS） carrying enhanced green fluorescent protein （EGFP） and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox-E1, 3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inos- shRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined. Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction （PCR） and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 10^11 VP/ml, 1.25 and 7.94 × 10^9 IU/ml, respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment. Key words: RNA activation; Inducible nitric oxide synthase; Adenovirus; EGFP