肿瘤抗原基因MAGE-3的克隆、原核表达与纯化

Cloning, prokaryotic expression and purification of tumor antigen gene MAGE-3

目的 :克隆人MAGE 3基因并进行原核表达和分离纯化 .方法 :通过RT PCR从人黑色素瘤细胞株中扩增MAGE 3全长cDNA ,经PCR扩增MAGE 3基因 3′端 32 4bp片段 ,克隆入pGEX 4T 1载体 ,构建重组原核表达质粒pGEX/MAGE 3,对含有该质粒的大肠杆菌DH5α进行诱导表达和分离纯化 .结果 :经RT PCR扩增出大小约 95 0bp的基因片段 ,以此为模板经PCR扩增出约 32 0bp片段 ,测序结果与Gen Bank公布的MAGE 3序列一致 ,成功构建了pGEX/MAGE 3原核表达质粒 ,经IPTG诱导在大肠杆菌中表达Mr 约 4 2 0 0 0的GST融合蛋白 ,纯化的蛋白纯度为 89% .结论 :成功克隆了人MAGE 3基因全长cDNA并对其 3′端 32 4bp片段编码的 1 0 8个氨基酸的蛋白进行了原核表达和分离纯化 。

AIM: To clone human MAGE 3 gene, to induce its prokaryotic expression and to purify the protein. METHODS: Full length cDNA of MAEG 3 gene was amplified by RT PCR from human melanoma cell line. 3′ terminal 324 bp segment of MAGE 3 gene was amplified by PCR. After the gene was sequenced, it was cloned into the expression vector pGEX 4T 1 to construct the expression plasmid pGEX/MAGE 3. The DH5αcontaining the expression plasmid was induced. RESULTS: The sequence of the amplified segment was identical with that reported in GenBank. The expression plasmid pGEX/MAGE 3 was successfully constructed. The DH5α containing the plasmid expressed a Mr 42 000 fusion protein after being induced by IPTG. Its purity was 89%. CONCLUSION: The full length cDNA of MAEG 3 gene was cloned. The 108 aa segment of MAGE 3 encoded by 3′ terminal 324 bp of gene is expressed and purified successfully, which lays a foundation for the application of the protein in tumor immunotherapy.