title: [Construction of MAGE-3 prokaryotic expression plasmid and its expression in Escherichia coli].
abstract: To express the GST-MAGE-3 protein in E. coli, and investigate the antitumor immune responses induced by Dendritic cells (DCs) pulsed with GST-MAGE-3 protein, the recombinant expression plasmid pGEX-MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR and confirmed by sequencing, and the pGEX-4T-2 vector. The recombinant plasmid was transformed into BL-21 E. coli. The expression of GST-MAGE-3 was induced with IPTG. The GST-MAGE-3 protein expressed as high as 32% of the total cellular protein. After purification with Glutathione Sepharose 4B, the purity of the protein was more than 90%, and 3mg GST-MAGE-3 was obtained from 100 mL BL-21 lysate. Dendritic cells from gastric carcinoma patients were pulsed with GST-MAGE-3 protein, and these DCs were used to stimulate the autologous T. lymphocytes. After 7 days, the T. lymphocytes cocultured with DCs pulsed with GST-MAGE-3 antigen exhibited specific cytotoxicity against MAGE-3 positive SGC-7901 cells. It is concluded that the GST-MAGE-3 protein are able to present antigen to T. lymphocytes, activate antigen-specific CTLs and induce special antitumor immune responses in vitro. Our results lay the groundwork for further research of the MAGE-3 vaccine.
authors.affiliation: Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Sun
authors.forename: Jin-Min
authors.initials: JM
authors.lastname: Wu
authors.forename: Xing-E
authors.initials: XE
authors.lastname: Liu
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D013274:Stomach Neoplasms / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins