Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 328-332, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-276119
ABSTRACT
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombination, Genetic
/
Recombinant Fusion Proteins
/
Transfection
/
Cell Line
/
Simplexvirus
/
Dependovirus
/
Brain-Derived Neurotrophic Factor
/
Genetic Vectors
/
Genetics
/
Metabolism
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2008
Type:
Article
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