Construction and expression of enhanced green fluorescent protein and MAGE-n co-expression vector

Objective:To construct the enhanced green fluorescent protein and MAGE-n co-expression vector and to express the plasmid in mouse melanoma B16 cells. Methods:The MAGE-n gene was obtained from pLXSN-MAGE-n by digestion, and subcloned into the multiclonal site of pIRES2-EGFP, then the pIRES2-EGFP-MAGE-n plasmid was constructed. The plasmid was transfected into the mouse melanoma B16 cells under mediation of lipofectamine. The positive clones were selected by G418. The expression of EGFP was detected by fluoresent microscope, and MAGE-n mRNA and protein in positive clones were detected by RT-PCR , Western blot and immunohistochemistry respectively. Results:The full ORF of MAGE-n, about 950bp fragment, was obtained from pLXSN-MAGE-n by enzyme digestion. The pIRES2-EGFP-MAGE-n plasmid was constructed and transfected into B16 cells successfully. Green fluorescence of EGFP expressed in B16 cells was observed under fluoresent microscope and it showed posotive signals of MAGE-n mRNA and protein in the transfected cells. Conclusion:The co-expression plasmid pIRES2-EGFP-MAGE-n was constructed successfully. The B16 cells that stably transfected MAGE-n was obtained which laid a foundation for the research of MAGE-n in the tumor immunotherapy.