Construction and identification of recombinant adenovirus with NT-3 gene

Objective:To construct the recombinant adenovirus vector carrying neurotrophin-3 (NT-3) gene.Methods:NT-3 and DsRed2 (including the ligation region pIRES2) fragment which had been digested by proper enzyme from NT-3-pIRES2-DsRed2 plasmid were cloned into adenovirus pAdShuttle-CMV plasmid.Adenovirus vector pAdShuttle-CMV-NT-3-DsRed2 were electroporated into BJ5183 which had been transformed into pAdEasy-1 previously to have the recombination with pAdEasy-1.Recombinant AdEasy plasmid DNA was analyzed and linearized by PacⅠ,then transfected into 293A cells with liposome method.The adenovirus that expressed NT-3 ptotein and red fluorescence was successfully constructed.The adenovirus was amplified in 293A cells three times,and high quality adenovirus was obtained by cesium chloride density gradient centrifugation.Western blot confirmed the expresseion of the NT-3 protein in 293T cells.Results:With the system of Adshuttle,a recombinant adenovirus expressing NT-3 protein and red fluorescence was successfully constructed.Conclusions:The recombinant adenovirus carrying NT-3 gene can be constructed with the system of Adeasy.