Construction of MAGE-3 prokaryotic expression plasmid and its expression in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 277-280, 2003.
Article
in Chinese
| WPRIM
| ID: wpr-270069
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pharmacology
/
Plasmids
/
Stomach Neoplasms
/
Dendritic Cells
/
T-Lymphocytes
/
Cells, Cultured
/
Blotting, Western
/
Cell Line, Tumor
/
Electrophoresis, Polyacrylamide Gel
/
Allergy and Immunology
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2003
Type:
Article
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