MAGE-A9基因原核表达系统的构建及其在肝细胞癌中的表达

目的扩增和克隆人黑色素瘤抗原MAGE-A9(melanom a antigen A9)cDNA,构建原核表达载体,制备抗MAGE-A9单抗,观察其在肝细胞癌中的定位。方法从人肝癌组织提取总RNA,用RT-PCR从中扩增出MAGE-A9cDNA,插入载体pMD18-T中,测序正确后,构建重组表达载体pBAD/gI-II-MAGE-A9,转化大肠杆菌TOP10株进行表达。重组蛋白经L-Arabinose诱导表达纯化后,制备抗MAGE-A9单抗,免疫组化检测MAGE-A9在肝细胞癌中的表达和定位。结果获得AGE-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-PAGE分析其相对分子质量为35Kd。获得抗MAGE-A9单抗,MAGE-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-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-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-A9可能成为肝癌特异性免疫治疗的靶点。

Construction of MAGE-A9 gene prokaryotic expression system and its expression in hepatocellular carcinoma

Objective It's 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.We 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 was expressed in a fair proportion of hepatocellular carcinoma specimens,these patients might be suitable candidates for immune involving antigen encoded by MAGE-A9 gene.