Cloning and expression of human melanoma antigen MAGEª²A10

    LI Lin, OUYANG Weiª²Ming, CHEN Liª²Hua, ZHUANG Ran, XIE Xin, FANG Liang, YANG Anª²Gang, JIN Boª²Quan

    Department of Immunology, School of Basic Medicine, Fourth Military Medical University, Xi¡¯an 710033, China

    ¡¾Abstract¡¿ AIM: To clone human MAGEª²A10 cDNA and to express its recombinant protein in E. coli. METHODS: The cDNA encoding human MAGEª²A10 gene was amplified by RTª²PCR from human melanoma cell line LiBr before the MAGEª²A10 gene was inserted into plasmids pMD18ª²T. After sequencing, the MAGEª²A10 was cloned into the prokaryotic expression vector pGEXª²4Tª²3 to construct the recombinant expression vector pGEXª²4Tª²3ª²MAGEª²A10 and was transformed into E. coli BL21. The recombinant GSTª²MAGEª²A10 fusion protein was expressed under induction of IPTG and GSTª²MAGE fusion protein was purified through glutathione agarose column. RESULTS: The sequence of MAGEª²A10 was identical to the sequence from GenBank. By affinity column and SDSª²PAGE, the purifiedª²GSTª²MAGEª²A10 fusion protein displayed a band of Mr67 000, and the antigenic specificity of the fusion protein was confirmed by Western blot. CONCLUSION: The MAGEª²A10 gene has been successfully cloned and expressed, which not only provides the immunogen for the preparation of antiª²MAGEª²A10 antibody but also is useful in further research on the mechanism of tumor pathogenesis and cellular immunological response to MAGE molecules. ......