Construction and identification of recombinant adenovirus vectors expressing GFP and human TERT-VEGF-Bcl-xl short hairpin RNA

Objective To construct and identify recombinant adenovirus vectors expressing EGFP and human TERT-VEGF-BCL-xl shRNA,and observe their transfection in human laryngocarcinoma cell line Hep-2. Methods Molecular biology techniques were used to subclone EGFP and human TERT-VEGF-Bcl-xl shRNA fragments from pGenesil-1.1 plasmid into the shuttle vector pENTR-EGFP-VTB. Then,the EGFP and human TERT-VEGF-Bcl-xl shRNA fragments were transformed to pGSadeno-EGFP-VTB using recombination in vitro. The linearized recombinant plasmid (pGSadeno-EGFP-VTB) by Pac Ⅰ was transfected into HEK293 cells to obtain recombinant adenovirus rAd-VTB. The recombinant adenovirus was identified by PCR,and the titer and transfection efficacy were evaluated by detecting the tag expression of EGFP. Results The results of enzymes digestion and PCR indicated that the recombinant adenovirus was correctly constructed. The titer was 1.0×109 PFU/ml,and the transfection efficacy in Hep-2 cell line was more than 90 percent. Conclusion The recombinant virus vector expressing EGFP and TERT-VEGF-Bcl-xl shRNA is successful constructed by using recombination in vitro.