Cloning of human melanoma antigen MAGE-A9 and its expression in hepatocellular carcinomas

ObjectiveTo express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens.MethodsMAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gIII to construct the recombinant expression vector pBAD/g III-MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay.ResultsThe cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 (21%) hepatocellular carcinoma specimens.ConclusionMAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.