Prokaryotic expression, purification and activity assay of recombinant vascular endothelial growth factor / 南方医科大学学报
Journal of Southern Medical University
;
(12): 1263-1268, 2006.
Artigo
em Inglês
| WPRIM
| ID: wpr-334947
ABSTRACT
<p><b>OBJECTIVE</b>To express human vascular endothelial growth factor (hVEGF(165)) in E. coli JM109 in the form of fusion protein by genetic engineering and test the biological activity and immunological competence of the expressed protein.</p><p><b>METHODS</b>hVEGF(165) gene was subcloned by PCR and inserted into pQE30 plasmid. hVEGF(165) fusion protein was expressed in E. coli JM109 and purified by Ni(2+)-NTA. The immunological competence of the expressed protein was tested by means of Western blotting and enzyme-linked immunosorbent assay (ELISA), and its biological activity was assayed by chicken chorioallantoic membrane (CAM) and Matrigel angiogenesis assay.</p><p><b>RESULTS</b>The recombinant hVEGF(165) fusion protein was successfully expressed in E. coli JM109 and its expression accounted for 30% of the total cellular protein. The purified protein presented a single band of 23 kD in SDS-PAGE. Western blotting, ELISA, CAM and matrigel angiogenesis assay showed excellent immunologic competence and biological activity of the recombinant protein.</p><p><b>CONCLUSION</b>Recombinant hVEGF(165) protein with excellent biological activity has been successfully expressed in E.coli JM109, which may facilitate future study in construction of prefabricated tissue-engineered bone graft.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Células Procarióticas
/
Proteínas Recombinantes
/
Ensaio de Imunoadsorção Enzimática
/
Western Blotting
/
Clonagem Molecular
/
Fator A de Crescimento do Endotélio Vascular
/
Escherichia coli
/
Genética
/
Metabolismo
Limite:
Humanos
Idioma:
Inglês
Revista:
Journal of Southern Medical University
Ano de publicação:
2006
Tipo de documento:
Artigo
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