Construction of recombinant GST-RCAS1 fusion gene and its expression in E. Coli / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences
; (6): 377-383, 2006.
Article
em Zh
| WPRIM
| ID: wpr-332139
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function.</p><p><b>METHODS</b>RCAS1 encoding gene was amplified by RT-PCR from total RNA extract of MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases BamH I and EcoR I. Then the ligated products were inserted into competence JM109 E. Coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. Coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-RCAS1 (N-18) and anti-RCAS1 (C-20) polyclonal antibody. The apoptosis of activated T cells induced by GST-RCAS1 fusion protein was detected by flow cytometry with Annexin V and propidium iodide (PI) staining.</p><p><b>RESULT</b>A 642 bp product was cloned by RT-PCR and the recombinant plasmid was constructed successfully. The GST-RCAS1 fusion protein was recognized by GST monoclonal antibody and RCAS1(N-18 and C-20) polyclonal antibody. FACS analysis showed that GST-RCAS1 fusion protein induced apoptosis in activated T cells.</p><p><b>CONCLUSION</b>The recombinant plasmid of RCAS1 has been successfully constructed and the GST-RCAS1 fusion protein expressed and purified. The apoptosis inducing effect of GST-RCAS1 fusion protein on activated T cells is demonstrated.</p>
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Assunto principal:
Plasmídeos
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Proteínas Recombinantes de Fusão
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Neoplasias da Mama
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Dados de Sequência Molecular
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Células Tumorais Cultivadas
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Sequência de Bases
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Expressão Gênica
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Alergia e Imunologia
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Escherichia coli
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Genética
Limite:
Humans
Idioma:
Zh
Revista:
Journal of Zhejiang University. Medical sciences
Ano de publicação:
2006
Tipo de documento:
Article