[Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor].
A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.
Li
Huiming
H
Central Laboratory, First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
Qiu
Wei
W
Wang
Feng
F
Wei
Fang
F
Chen
Xiafang
X
Wu
Xiaobing
X
Huang
Qian
Q
D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000235:genetics; D014162:Transfection
D019208:Brain-Derived Neurotrophic Factor; D011993:Recombinant Fusion Proteins