OBJECTIVETo investigate the rapid construction of enhanced green fluorescent protein (EGFP) labeled recombinant adenovirus containing hVEGF165 and its distribution and efficiency in bone marrow transplanted mice.

METHODSThe recombinant adenovirus Ad-EGFP/hVEGF165 was rapidly constructed by using AdEasy system based on the homologous recombination in bacteria, and its property was studied in vitro. Then 3x10(8) PFU adenovirus was injected into BALB/c mice via the tail vein accepted syngeneic bone marrow transplantation. The in vivo distribution of adenovirus and plasma levels of VEGF were measured at different phases.

RESULTSThe adenovirus Ad-EGFP/hVEGF165 was quickly constructed by homologous recombination in bacteria using AdEasy system. The purified particles were homogenous hexagon with titers between 10(10) PFU/ml and 10(11) PFU/ml. The Hela cells infected with Ad-EGFP/hVEGF165 did not show cytopathic effects after several times passages. Under the fluorescent microscope, EGFP was revealed in the heart, lung, liver, spleen, kidney and intestine of mice at different phases. RT-PCR and immunohistochemistry showed hVEGF165 expressed significantly. No obvious damages were observed in different organs by HE staining. The plasma level of hVEGF165 was up to 866.67+/-97.13 pg/ml.

CONCLUSIONThese results suggested that the construction of adenovirus vector by homologous recombination in bacteria was an efficient and time-saving method, and high titer adenovirus could successfully mediate the safe and stable expression of hVEGF165 in post bone marrow transplanted mice. All these would make further gene therapy in bone marrow transplantation possible.