[Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor]

Sheng Wu Gong Cheng Xue Bao. 2008 Feb;24(2):328-32.
[Article in 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.

Publication types

  • English Abstract
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain-Derived Neurotrophic Factor / biosynthesis*
  • Brain-Derived Neurotrophic Factor / genetics
  • Cell Line
  • Dependovirus / genetics
  • Dependovirus / metabolism*
  • Genetic Vectors / genetics*
  • Humans
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Recombination, Genetic
  • Simplexvirus / genetics
  • Transfection

Substances

  • Brain-Derived Neurotrophic Factor
  • Recombinant Fusion Proteins