Construction and Expression of Bicistronic Adenovirus Vector Carrying Thymidine Kinase and Enhanced Green Fluorescent Protein Gene.

Objective:To construct a recombinant adenovirus vector carrying gene of herps simplex virus thymidine kinase(HSV-tk) and enhanced green fluorescent protein(EGFP),and detect its expression in rat endothelial cells.Methods: Molecular cloning techniques were used to construct an adenovirus vector.The internal ribosome entry site(IRES) of encephalomyocarditis virus(EMCV),which could coordinate expression of two genes in a single vector,was optioned.The recombinant advenovirus vector was obtained by cotransforming pShuttleCMV-tk-EGFP and adenovirus genomic DNA plasmid of pAdeasy-1 vector in bacterial cells.Then the vector containing tk-IRES-EGFP was transfected into rat endothelial cells(RECs).Results: An adenovirus vector carrying tk-IRES-EGFP was constructed.The fluorescence microscope findings showed that the tk-IRES-EGFP gene was successfully transferred into RECs.There were no differences in the growth pattern or the morphology between RECs and REC/ tk-IRES-EGFP cells.In vitro experiments demonstrated dose-dependent cell killing by transduction of tk-IRES-EGFP gene followed by Ganciclovir(GCV) treatment.Conclusion: These results suggest that this new kind of adenovirus vector can serves as a new tool and method for tumor anti-angiogenic gene therapy.