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

来源 :Journal of Nanjing Medical University | 被引量 : 0次 | 上传用户:wuzhen16885168
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Objective:To 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.Methods:MAGE-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/gⅢto construct the recombinant expression vector pBAD/gⅢ- MAGE-A9,and was transformed into E.coil 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.Results:The 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. Conclusion:MAGE-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. Objective: To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods: MAGE-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 / gIIIto construct the recombinant expression vector pBAD / gIII- MAGE-A9, and was transformed into 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 was was through ABC assay. Results: The 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, 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. Conclusion: MAGE- 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.
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