Construction and identification of replication recombinent adenovirus vector of human TGF-β1 shRNA

Abstract

Objective To construct the recombinant adenovirus vector with human gene TGF-β1 (shRNA,short hairpin RNA)by Cre/lox P system.Methods Human U6 promoters following TGF-β1 shRNA(U6shTGF-β1)were obtained with PCR.Then the promoters were ligated to T-vector.After plasmids pT-U6shTGF-β1 being formed.these plasmids were transformed into E.coli JM109.The target gene fragment was subcloned into a shuttle plasmid PDC316 to construct recombinant shuttle plasmid PDC3 16-TGF-β1.Then the combinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1 and 3Cre were cotransfected into 293T cell to construct recombinant adenovirus AdTGF-β1,which was then identified by infection test and PCR amplification.Results U6shTGF-β1 was obtained.The sequence of cloned target fragment was completely consistent with designed sequence.After purification and concentration,the titer of AdTGF-β1 reached 107.3 TCID50/ml.Ad TGF-β1 could transfect 293T cell.Both adenovirus and TGF-β1 target gene fragment could be amplified from rAdTGF-β1 by PCR.Conclusions The recombinant adenovirus expression vector of TGF-β1 and control gene are successfully constructed.This study establishes a foundation for further study on TGF-β1 RNAi vaccines and new gene therapy for human keloid and hypertrophic scar. Key words: Transforming growth factor-β1; Adenovirus vector; RNA interference; Gene therapy; Cicatrix