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 1x10(12) and 8x10(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.
Chen
Qian
Q
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. fanm@nic.bmi.ac.cn
Wang
Jia-Zheng
JZ
Liu
Hong
H
Wu
Yan
Y
Fan
Ming
M