人黑色素瘤抗原MAGE-A10的基因克隆及原核表达

Cloning and expression of human melanoma antigen MAGE-A10

目的 :扩增和克隆人黑色素瘤抗原MAGE A1 0(melanomaantigenA1 0 )cDNA ,构建原核表达载体并在大肠杆菌表达 .方法 :从人黑色素瘤细胞系LiBr中提取总RNA ,用RT PCR从中扩增出MAGE A1 0cDNA ,插入载体pMD1 8 T中 .测序正确后 ,克隆至原核表达载体pGEX 4T 3中 ,构建重组表达载体pGEX 4T 3 MAGE A1 0 ,转化大肠杆菌BL2 1株进行表达 .经IPTG诱导表达 ,谷胱甘肽亲和层析获得重组目的蛋白 .结果 :成功获得MAGE A1 0编码基因并构建原核表达载体 ,测序结果与GenBank收录序列相一致 .可以获得部分可溶性的原核重组蛋白 ,经亲和层析和分析 ,证实融合蛋白占总量的 5 8.9% .SDS PAGE分析其相对分子质量 (Mr)为 6 70 0 0 ,Westernblot证实为目的蛋白 .结论 :成功获得MAGE A1 0cDNA ,构建得原核表达载体并获得高效表达 .本研究为制备MAGE A1 0mAb及研究MAGE A1 0可能参与肿瘤发生发展的机制奠定了基础 .

AIM: To clone human MAGE A10 cDNA and to express its recombinant protein in E. coli . METHODS: The cDNA encoding human MAGE A10 gene was amplified by RT PCR from human melanoma cell line LiBr before the MAGE A10 gene was inserted into plasmids pMD18 T. After sequencing, the MAGE A10 was cloned into the prokaryotic expression vector pGEX 4T 3 to construct the recombinant expression vector pGEX 4T 3 MAGE A10 and was transformed into E. coli BL21. The recombinant GST MAGE A10 fusion protein was expressed under induction of IPTG and GST MAGE fusion protein was purified through glutathione agarose column. RESULTS: The sequence of MAGE A10 was identical to the sequence from GenBank. By affinity column and SDS PAGE, the purified GST MAGE A10 fusion protein displayed a band of M r 67 000 , and the antigenic specificity of the fusion protein was confirmed by Western blot. CONCLUSION: The MAGE A10 gene has been successfully cloned and expressed, which not only provides the immunogen for the preparation of anti MAGE A10 antibody but also is useful in further research on the mechanism of tumor pathogenesis and cellular immunological response to MAGE molecules.