Construction and characterization of recombinant adenoviruses expressing biologically active human brain-derived neurotrophic factor and neurotrophin-3

In order to deliver brain-derived neurotrophic factor(BDNF) and neurotrophin-3(NT3) into CNS to prevent or reduce degeneration of CNS neurons in human neurodegenerative diseases and neuron injuries, recombinant adenoviruses Ad-BDNF and Ad-NT3 were constructed. BDNF and NT3 genes were inserted into an E1-substituted adenovirus shuttle plasmid, respectively, and were driven by the human cytomegalovirus immediate-early gene promoter/enhancer. After cotransfection into 293 cells with the adenovirus plasmid pJM17, the recombinant adenovirus Ad-BDNF and Ad-NT3 were propagated in 293 cells via homologous recombination. After two rounds of CsCl centrifugation, the human recombinant adenovirus Ad-BDNF and Ad-NT3 were obtained with titer of 1 X 10(12) and 8 x 10(11) pfu/ml, respectively. To examine the expression of BDNF and NT3, HeLa cells were infected with Ad-BDNF and Ad-NT3, respectively. RT-PCR, Western blot and ELISA analysis results showed that BDNF and NT3 genes could be transcribed and translated in Ad-BDNF and Ad-NT3 infected HeLa cells. And the culture media containing 10% conditioned medium of Ad-BDNF and Ad-NT3 infected Hela cells could induce the neurite outgrowth from E8 dorsal root ganglion neurons.