Construction and identification of recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor short hairpin RNA

Objective To construct and identify recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor (VEGF) iRNA and to inhibit the gene expression of VEGF in human nasopharyngeal carcinoma cell line CNE with the RNA interference (RNAi) technique,and explore the expression of VEGF in CNE and its proliferation before and after transfection.Methods By the method of restriction endonuclease digestion,the GFP and human VEGF genes were subcloned from pGenesil-1 plasmid into the shuttle vector pGSadeno-GFP-VEGF.After correct identification of the recombinant plasmid pGSadeno-GFP-VEGF,it was then digested by Pacl and transfected into HEK293 cells to be packaged and amplification for rAd-VEGF.Meanwhile,the recombinant adenovirus was identified by PCR method.The expression of GFP was detected to evaluate the titre and transfective ratio.Results Digestion and PCR identification showed the construction of recombinant adenovirus rAd-VEGF was successful via LR recombinant.The titre was 2×109 PFU/ml while the transfective ratio in CNE cell line was more than 90 percent which met the need of experiment.Cell growth was inhibited compared with non-siRNA transfected CNE cell line.Conclusion The recombinant virus expressing GFP and VEGF are successfully constructed; it layed a foundation for further relevant study. Key words: Nasopharyngeal neoplasms;  Vascular endothelial growth factors;  RNA interference; Adenoviridae