Construction Of Recombinant Adenovirus Vector Expressing Platelet-derived Growth Factor C And Its Expression In Ecps

Objective: To construct the recombinant adenovirus vector whichencode rat PDGF-C gene and to observe the expression of target gene inendothelial progenitor cells derived from rat bone marrow.Methods: Through RT-PCR, the gene which encode rat PDGF-C wasobtained from pIRES-EGFP-PDGF-C. The gene fragment was cloned intopDONR221vector via BP recombination, and then subcloned intopAd/CMV/V5-DEST gateway vector via LR recombination. Afterconfirmed by PCR and DNA sequencing, the adenovirus vector whichcarried PDGF-C gene was linearized with PacI enzyme and transfected intoHEK293A cells. They packaged recombinant adenovirus particles. The titreof Ad-PDGF-C was measured with the help of expression of enhancedgreen fluorescent protein (EGFP). EPCs was derived from rat bone marrowand obtained by adherent culture. The recombinant adenovirus wastransfected into EPCs. The expression of PDGF-C was detected byReal-time qPCR and Western blot analysis respectively．Results: The evidence of endonuclease digestion and PCR analysisconfirmed that PDGF-C gene was correctly inserted into thepAD/CMV/V5-DEST gateway vector. After transfection, of PDGF-Cprotein in pAD-PDGF-C was expressing significantly higher than thatbefore transfected EPCs group and that of transfected with Ad-GFP.Conclusion: The recombinant adenovirus vector which rat PDGF-Ccontained was constructed correctly, which can mediate target gene expression in transfected EPCs effectively. This study provides a researchbasis for constructing genetic transfection tool in follow-up gene therapystudy．...