Construction of recombinant adenoviruse vector containing human vascular endothelial growth factor and its expression in mesenchymal stem cells

Objective To construct the adenoviruse vector containing human VEGF_ 165 and to investigate the property of the transfected bone mesenchymal stem cells in vitro.Methods VEGF_ 165 cDNA was cloned into the shuttle plasmid pShuttle-CMV,then pShuttle-VEGF_ 165 was linearized and cotransferred with adenoviral backbone vector into E.coli strain BJ5183.The recombinant Ad plasmid was homologously recombinated in bacterial cells.After screening and amplification,the recombinant Ad plasmid was digested with Pac I and transfected into HEK293 cells.The replication-defective adenovirus Ad-VEGF_ 165 was packed and amplified in the HEK293 cells.The rat MSCs were transfected by the adenovirus and the expression of VEGF_ 165 gene was detected.Results Restriction endonuclease analysis and gene sequencing confirmed that the recombinant contained VEGF_ 165 cDNA.Under electron microscopy,the viral particles were easily detected in HEK293 cells.Virus titer could reach as high as 6.3×10 10 TCID50/ml.After the MSCs were transfected by the virus,RT-PCR,immunohistochemistry and Western blot revealed that VEGF_ 165 was transcripted and expressed in transgene MSCs.Conclusion The construction of recombinant adenovirus vector by homologous recombination in bacterial cells can be quickly and easily performed.The VEGF_ 165 gene mediated by adenovirus with a high infectivity is efficient for gene therapy of MSCs line.