MAGEA9基因原核表达系统构建及其在肝细胞癌中表达.docVIP

MAGEA9基因原核表达系统构建及其在肝细胞癌中表达 　　【摘要】 目的 扩增和克隆人黑色素瘤抗原MAGE-A9(melanoma 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-A9 cDNA,插入载体pMD18-T中，测序正确后,构建重组表达载体pBAD/gIII-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分析其相对分子质量为35 Kd。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。 　　【关键词】 黑色素瘤抗原；MAGE-A9；RT-PCR；基因克隆；基因表达 　　 　　Construction of MAGE-A9 gene prokaryotic expression system and its expression in hepatocellular carcinoma 　　XU Lu，ZHU Jin，QIU Zhen-ning, et al. Department of Pathology, Nanjing Medical University, Nanjing 210029, China 　　 　　【Abstract】 　　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, 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 specimen