Construction of MAGE-3 prokaryotic expression plasmid and its expression in Escherichia coli.
- Author:
Xiao-Dong SUN
1
;
Jin-Min WU
;
Xing-E LIU
Author Information
1. Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China.
- Publication Type:Journal Article
- MeSH:
Antigens, Neoplasm;
genetics;
isolation & purification;
metabolism;
pharmacology;
Blotting, Western;
Cell Line, Tumor;
Cells, Cultured;
Dendritic Cells;
drug effects;
immunology;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Neoplasm Proteins;
genetics;
isolation & purification;
metabolism;
pharmacology;
Plasmids;
genetics;
Stomach Neoplasms;
immunology;
T-Lymphocytes;
immunology
- From:
Chinese Journal of Biotechnology
2003;19(3):277-280
- CountryChina
- Language:Chinese
-
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.
