[Construction of recombinant GST-RCAS1 fusion gene and its expression in E. Coli].
To construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function.



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.



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.



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.
Hong
Xue-jun
XJ
Institute of Immunology, Zhejiang University, Hangzhou 310031, China.
Shen
Fen-ping
FP
Wang
Qing-qing
QQ
D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; C120762:EBAG9 protein, human; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase