Construction of VEGF recombinant plasmid pcDNA/V and its expression in model rats with acute myocardial ischemia / 生物工程学报
Chinese Journal of Biotechnology
; (12): 220-225, 2006.
Article
em Zh
| WPRIM
| ID: wpr-286305
Biblioteca responsável:
WPRO
ABSTRACT
The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Patologia
/
Terapêutica
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Proteínas Recombinantes
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Transfecção
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Terapia Genética
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Distribuição Aleatória
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Linhagem Celular
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Galinhas
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Ratos Sprague-Dawley
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Fator A de Crescimento do Endotélio Vascular
Tipo de estudo:
Clinical_trials
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Prognostic_studies
Limite:
Animals
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Humans
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Male
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2006
Tipo de documento:
Article