Construction of hVEGF165 Recombinant Adenovirus Vector and Its Expression in HEK293 Cells

Objective: To construct human vascular endothelial growth factor165(hVEGF165) recombinant adenovirus and investigate its expression in HEK293.Methods: The target gene of human VEGF165 was obtained from plasmid pcDNA3/hVEGF165 and was verified by DNA sequence analysis.The hVEGF165 gene was subcloned into shuttle vector pAdTrack-CMV.After identified with restriction enzymes,plasmid pAdTrack-hVEGF165 was linearized by digestion with restriction endonuclease PmeⅠ,and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination.After linearized by PacⅠ,the homologous recombinant adenovirus plasmid was transfected into HEK293 cells recombinant adenovirus.The high-level adenoviruses infected HEK293 cells and the expression of VEGF-1 by the transfected BMSC was examined with RT-PCR assay and ELISA.Results: The recombinant plasmid pAd-hVEGF165 was subjected to sequence analysis which indicated all nucleotides were identical to the human VEGF165 sequence provided by Genbank.After transfection,the expression of VEGF165 in HEK293 cell was detected.Conclusion: Combinant plasmid pAd-hVEGF165 was constructed in vitro and expressed successfully in HEK293 cells.