Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines.
Biologicals
; 37(1): 37-43, 2009 Jan.
Article
em En
| MEDLINE
| ID: mdl-19008122
ABSTRACT
A novel plasmid pGS/2SS-M4GFP was constructed in the present study by recombination of GS/2SS gene and enhanced green fluorescent protein (M4GFP) sequence. The GS/2SS fusion gene encoding two copies of somatostatin genes was firstly introduced into pVAX-asd vector in which the kanamycin resistance cassette was replaced by the asd cassette. The M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame. After purified, plasmid pGS/2SS-M4GFP was transfected into different cell lines derived from pig kidney and human cancer cells. The transcription process of GS/2SS gene was confirmed by RT-PCR, and the localization as well as expression of GS/2SS-M4GFP fusion protein was observed by confocal microscopy and ELISA. Transfection results revealed that sole M4GFP was localized within the cytosol and the nucleus, while fusion protein GS/2SS-M4GFP was localized only in the cytoplasm. Furthermore, it should be noted that subcellular localization of GS/2SS-M4GFP was not specific to one cell line, but appeared to be common across a variety of cell lines. These results provide for the first time valuable evidence that M4GFP is a versatile tool to trace GS/2SS protein and pave the way for further study on its tissue distribution and immunological mechanism in vivo.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Linhagem Celular
/
Transgenes
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Proteínas de Fluorescência Verde
/
Vetores Genéticos
Tipo de estudo:
Evaluation_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article