Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor.
- Author:
Huiming LI
1
;
Wei QIU
;
Feng WANG
;
Fang WEI
;
Xiafang CHEN
;
Xiaobing WU
;
Qian HUANG
Author Information
1. Central Laboratory, First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
- Publication Type:Journal Article
- MeSH:
Brain-Derived Neurotrophic Factor;
biosynthesis;
genetics;
Cell Line;
Dependovirus;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Recombination, Genetic;
Simplexvirus;
genetics;
Transfection
- From:
Chinese Journal of Biotechnology
2008;24(2):328-332
- CountryChina
- Language:Chinese
-
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.
