【摘要】 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。更多还原

【Abstract】 Objective: To clone human MAGE-A9 cDNA, to express its recombinant protein in E.coli and to examine the expression of MAGE-A9 in hepatocellular carcinoma specimens. Methods: The cDNA encoding human MAGE-A9 gene was amplified by RT-PCR from human hepatocelluar carcinoma tissue before the MAGE-A9 gene was inserted into plasmids pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ-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. Results: The sequence of MAGE-A9 was identical to the sequence from GenBank. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35 000. Anti-MAGE-A9 monoclonal antibody was procuced. It was found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected on 8 cases out of 39 (21 %) hepatocellular carcinoma specimens. Conclusion: MAGE-A9 antigen is expressed in a fair proportion of hepatocellular carcinoma specimens. These patients might be suitable candidates for immune involving antigen encoded by MAGE-A9 gene.更多还原