Construction of recombinant GST-RCAS1 fusion gene and its expression in E. Coli.
- Author:
Xue-jun HONG
1
;
Fen-ping SHEN
;
Qing-qing WANG
Author Information
- Publication Type:Journal Article
- MeSH: Antigens, Neoplasm; biosynthesis; genetics; Base Sequence; Breast Neoplasms; genetics; immunology; Escherichia coli; genetics; metabolism; Gene Expression; Glutathione Transferase; biosynthesis; genetics; Humans; Molecular Sequence Data; Plasmids; genetics; Recombinant Fusion Proteins; biosynthesis; isolation & purification; Tumor Cells, Cultured
- From: Journal of Zhejiang University. Medical sciences 2006;35(4):377-383
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function.
METHODSRCAS1 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.
RESULTA 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.
CONCLUSIONThe 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.