Objective:The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research.Methods:The human DBC2 gene was first subcloned into a shuttle plasmid pAdTrack-CMV.After recombining with pAdEasy-1 vector in BJ5183 cells,the new recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus.The human bladder cancer cell line T24 was infected with DBC2-containing adenovirus particles.Both RNA and protein were collected from cells harvested at 72 h after infection.Real time quantitative PCR(qPCR) and Western blot were used to examine mRNA and protein levels.Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein.Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR.Pac I digest of the final produced recombinant vector yielded band sizes of approximately 30 kb and 4.5 kb.After virus infection with the pAdEasy-DBC2-CMV vector,the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope.qPCR and Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virus transfected cells.Conclusion:By using the pAdEasy adenovirus system,we successfully constructed an adenovirus that could highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line.This viral construct would be widely used for our further research in gene functional assays and gene therapy in bladder cancer. Objective: The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2 (deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research. Methods: The human DBC2 gene was first subcloned into a The plasmid pAdTrack-CMV. After recombining with pAdEasy-1 vector in BJ5183 cells, the new recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus. T24 was infected with DBC2-containing adenovirus particles.Both RNA and protein were collected from cells harvested at 72 h after infection. Real time quantitative PCR (qPCR) and Western blot were used to examine mRNA and protein levels. Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein . Results: Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR. Pac I digest of the final produced recombinant vector yielded band sizes of approximately 30 k b and 4.5 kb. After virus infection with the pAdEasy-DBC2-CMV vector, the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope. qPCR and Western blot assay that the DBC2 gene was overexpressed at both the mRNA and protein levels in virus transfected cells. Conlusion: By using the pAdEasy adenovirus system, we successfully constructed an adenovirus that could highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line. This viral construct would be widely used for our further research in gene functional assays and gene therapy in bladder cancer.