〔Abstract〕 Objective To construct eukaryotic expression plasmid encoding MAGE-A3, preparein vitrotranscribed mRNA and express MAGE-A3 protein in NIH/3 T3 cells. Methods The whole gene of MAGE-A3 was amplified by RT-PCR and cloned into pcDNA3.1(+) expression vector to build pcDNA3.1-MAGE. Subsequently, the template DNA for transcriptionin vitrowas prepared by PCR.Then, the template DNA was transcribed into MAGE-A3 RNAs using MEGAscript T7 transcription kit. The transcripts were added a poly(A) tail to the 3' termini and purified by using transcription clean-up kit. After transfection of the purified MAGE-A3 mRNA into NIH/3 T3 cells, Western blot analysis was used to confirm the expression of MAGE-A3 protein. Results The gene inserted into the recombinant vector was proven to be completely identical with the sequence of the MAGE-A3 gene in online database. The ratio of A260/A280of the purified mRNA was 1.92, and the concentration was 872.3 ng/μl. The length of MAGE-A3 mRNA was about 500 bp. After MAGE-A3 mRNA transfected into NIH/3 T3 cells, MAGE-A3 protein could be produced in cells and the size of protein was 35 ku. Conclusion Eukaryotic expression plasmid encoding MAGE-A3 is successfully constructed and the high-purity MAGE-A3 mRNA is obtainedin vitro. The mRNA can be translated into MAGE-A3 protein in NIH/3 T3 cells.