Construction of eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 WT or its variants / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences
; (6): 189-194, 2011.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-319811
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its variants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells.</p><p><b>METHODS</b>The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+). pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs (W39R, R264C, W39R-R264C). pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells.</p><p><b>RESULT</b>The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing. pcDNA3.1(+)-CYP19(W39R)-GFP, pcDNA3.1(+)-CYP19(R264C)-GFP, pcDNA3.1(+)- CYP19(W39R-R264C)-GFP plasmids were confirmed by DNA sequencing. The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully, which lays the foundation for the research of biological activities of CYP19 variant allozymes.</p>
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Proteínas Recombinantes de Fusão
/
Aromatase
/
Transfecção
/
Mutagênese Sítio-Dirigida
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Linhagem Celular Tumoral
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Proteínas de Fluorescência Verde
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Vetores Genéticos
/
Genética
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Zhejiang University. Medical sciences
Ano de publicação:
2011
Tipo de documento:
Artigo