Construction of MAGE--3 gene eukaryotic expression vector and its expression in mouse melanoma cells

Objective To amplify human melanoma antigen 3 (MAGE-3) gene and construct the eukaryotic expression vector that can be expressed in B16 mouse melanoma cells. Methods The MAGE-3 gene was amplified by PCR, then cloned into the vector pIRES2-EGFP for constructing the pIRES2-EGFP-MAGE-3 plasmid. The plasmid was transfected into the B16 mouse melanoma cells under mediation of lipofectamine and the positive clones were selected. The expression of enhanced green fluorescent protein (EGFP) and MAGE-3 mRNA in positive clones were detected by fluorescence microscopy and RT-PCR, respectively. Results The full length of MAGE-3 was amplified by PCR. The pIRES2-EGFP-MAGE-3 plasmid was constructed and transfected into B16 cell successfully. Green fluorescence of fused protein expression was found and MAGE-3 mRNA expressions were detected in the positive clones. Conclusion The eukaryotic expression plasmid pIRES2-EGFP-MAGE-3 was constructed successfully. The B16 cell lines that stably transfected MAGE-3 is obtained, which contribute to the research of MAGE-3 in the immunotherapy of tumor.