Construction and
Characterization of Recombinant Adenoviruses Expressing Biologically Active
Human Brain-derived Neurotrophic Factor and Neurotrophin-3
CHEN Qian, WANG Jia-Zheng, LIU Hong, WU Yan, FAN Ming
( Institute of Basic Medical Sciences, Academy of Military Medical
Sciences, Beijing 100850, China )
Abstract 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 1×1012
and 8×1011 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.
Key words BDNF； NT3； recombinant adenovirus； gene expression； biological activity
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