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目的:扩增和克隆人黑色素瘤抗原MAGE鄄A9(melanomaantigenA9)cDNA,构建原核表达载体,制备抗MAGE鄄A9单抗,观察其在肝细胞癌中的表达。方法:从人肝癌组织提取总RNA,用RT-PCR从中扩增出MAGE鄄A9cDNA,插入载体pMD18鄄T中,测序正确后,构建重组表达载体pBAD/gIII鄄MAGE鄄A9,转化大肠杆菌TOP10株进行表达。重组蛋白经L鄄Arabinose诱导表达纯化后,制备抗MAGE鄄A9单抗,免疫组化检测MAGE鄄A9在肝细胞癌中的表达和定位。结果:获得MAGE鄄A9cDNA,测序结果与GenBank一致。成功构建表达载体,表达可溶重组蛋白,SDS鄄PAGE分析其相对分子质量为35kD。获得抗MAGE鄄A9单抗,MAGE鄄A9在肝细胞癌中的阳性表达率为21%(8/39),主要表达于胞浆,未见肿瘤异质性,统计学分析MAGE鄄A9的表达与患者年龄、性别、肿瘤大小和分化程度没有相关性。结论:有相当部分肝癌患者的肿瘤表达MAGE鄄A9抗原,且其表达与患者年龄、性别、肿瘤大小和分化程度没有相关性,MAGE鄄A9可能成为肝癌特异性免疫治疗的靶点。
OBJECTIVE: To amplify and clone the cDNA of melanomaantigen A9, and to construct the prokaryotic expression vector to prepare anti-MAGE-A9 monoclonal antibody to observe its expression in hepatocellular carcinoma. Methods: Total RNA was extracted from human hepatocellular carcinoma tissue. MAGE-A9 cDNA was amplified by RT-PCR and inserted into vector pMD18-T. After sequencing, the recombinant plasmid pBAD / gIII-MAGE-A9 was constructed and transformed into E.coli TOP10 Express. The recombinant protein was induced by L-Arabinose and purified to prepare anti-MAGE-A9 monoclonal antibody. The expression and localization of MAGE-A9 in hepatocellular carcinoma were detected by immunohistochemistry. Results: MAGE-A9 cDNA was obtained and the sequencing result was consistent with GenBank. The expression vector was successfully constructed and the soluble recombinant protein was expressed. The relative molecular mass was 35kD by SDS-PAGE analysis. MAGE-A9 monoclonal antibody was obtained. The positive expression rate of MAGE-A9 in hepatocellular carcinoma was 21% (8/39), mainly expressed in the cytoplasm. No tumor heterogeneity was found. The expression of MAGE-A9 There was no correlation with patient’s age, sex, tumor size and degree of differentiation. Conclusion: MAGE-A9 antigen was expressed in the tumor of a considerable number of patients with HCC, and its expression was not related to the age, sex, tumor size and differentiation. MAGE-A9 may be the target of HCC-specific immunotherapy.