Construction and identification of recombinant adenoviral vector co-expressing connexon 43 and green fluorescent protein

Objective To construct and identify the recombinant adenoviral vector co-expressing connexon 43 and green fluorescent protein.Methods The Cx43 gene was amplified by PCR from eukaryotic expression vector pcDNA3.0-Cx43 and cloned into the shuttle plasmid pAdTrack-CMV which expressed the report gene GFP.Then the recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l.The recombinant plasmid pAd-Cx43-GFP was confirmed with single digestion by PacⅠ.Further,the plasmid pAd-Cx43-GFP was transfected into 293 cells,and the expression of Cx43 and GFP in 293 cells were respectively detected by fluorescent microscopy and Western blot.Results(1)The single fragment which length approached to Cx43 was obtained using PCR;(2)Double digested with restriction endonucleases NotⅠ/XhoⅠ,the recombinant shuttle plasmid pAdTrack-Cx43 was confirmed by two products which length were respectively about 9kb and 1kb;(3)Two fragments of about 31kb and 4kb were obtained,after the recombinant plasmid pAd-Cx43-GFP was digested with PacⅠ,which suggested a successful homologous recombination;(4)After transfected by pAd-Cx43-GFP,the expression of GFP was monitored by fluorescent microscopy in 293 cells;(5)In contrast to no-transfection group,higher expression of Cx43 in 293 cells transfected by pAd-Cx43-GFP was detected by Western blot.Conclusion The recombinant adenoviral vector Ad-Cx43-GFP was successfully constructed.