肿瘤抗原基因MAGE-A9的克隆及原核表达

Cloning and prokaryotic expression of tumor antigen MAGE-A9

目的扩增和克隆人黑色素瘤抗原MAGE-A9cDNA,构建原核表达载体,表达并纯化MAGE-A9蛋白。方法从人肝癌组织提取总RNA,用反转录-聚合酶链反应(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。以L-Arabi-nose进行诱导表达,并纯化。结果获得MAGE-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,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析其相对分子质量为35 000。结论成功地构建了pBAD/gⅢ-MAGE-A9原核表达质粒,获得了MAGE-A9蛋白,为该蛋白应用于肿瘤免疫治疗奠定了基础。

Objective To amplify MAGE-A9 gene and construct the recombinant prokaryotic plasmid to induce expression of MAGE-A9. 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/g Ⅲ to 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-Arabineae and was purified through Hitrap column. Results The sequence of MAGE-A9 was identical to the sequence from C, enBank. The expression plasmid pBAD/g Ⅲ-MAGE-A9 was successfully constructed, By affinity column and SDS-PAGE,the purified MAGE-A9 fusion protein displayed a band of Mr 35 000. Conclusion The expression plasmid pBAD/gⅢ-MAGE-A9 was successfully constructed. MAGE-A9 was expressed and pufffled successfully, which may lay foundation for application of the protein in tumor immunotherapy.