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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.
Artigo em Chinês | 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.
Assuntos
Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Recombinação Genética / Proteínas Recombinantes de Fusão / Transfecção / Linhagem Celular / Simplexvirus / Dependovirus / Fator Neurotrófico Derivado do Encéfalo / Vetores Genéticos / Genética / Metabolismo Limite: Humanos Idioma: Chinês Revista: Chinese Journal of Biotechnology Ano de publicação: 2008 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Recombinação Genética / Proteínas Recombinantes de Fusão / Transfecção / Linhagem Celular / Simplexvirus / Dependovirus / Fator Neurotrófico Derivado do Encéfalo / Vetores Genéticos / Genética / Metabolismo Limite: Humanos Idioma: Chinês Revista: Chinese Journal of Biotechnology Ano de publicação: 2008 Tipo de documento: Artigo