Construction of recombinant adenovirus vector containing transcriptional factor activator protein 2α and its expression in rat mesenchymal stem cells

Objective To construct the recombinant adenovirus vector containing transcriptional factor activator protein(AP) 2α,infect rat mesenchymal stem cells(MSCs) and determine its expression.Methods AP2α gene was amplified from the total RNA of rat PC12 cells by PCR and cloned into adenovirus shuttle plasmid pAdtrace-TOX,and the constructed recombinant plasmid pAdtrace-AP2α was subjected to homologous combination with adenovirus skeleton plasmid pAd-Easy1 in E.coli BJ5183.The obtained recombinant adenovirus vector pAd-AP2α was transfected to HEK293 cells for packaging.Rat MSCs were infected with the prepared recombinant adenovirus Ad-AP2α,then observed for expression of RFP by fluorescent microscopy,and determined for expression of AP2α by real-time PCR and Western blot.Results The target gene band at a length of about 1 400 bp was observed in recombinant plasmid pAdtrace-AP2α by PCR and restriction analysis,of which the sequence was consistent with that reported in GenBank,and was cloned into adenovirus vector correctly.Recombinant adenovirus vector pAd-AP2α was successfully packaged in HEK293 cells,and the obtained recombinant adenovirus Ad-AP2α reached a titer of 2.6 × 108 pfu / ml.More than 60% of MSCs were positive for RFP 48 h after infection.Real-time PCR and Western blot showed that the expression level of AP2α increased significantly 72 h after infection(P 0.01).Conclusion Recombinant adenovirus vector containing AP2α was successfully constructed,which infected MSCs effectively and enhanced the expression of AP2α in MSCs.