人睾丸肿瘤抗原MAGE-E1基因的克隆测序及在原核系统中的表达

目的：扩增及克隆人的MAGE-E1基因并利用大肠杆菌进行表达。方法：从人胶质瘤细胞系BT-325中提取总RNA，用RT-PCR从中扩增出MAGE-E1基因片段。将MAGE-E1基因片段插入载体pGEM-T easy中，经全自动序列分析仪测序正确后，再克隆至表达载体pGEX-4T-2中，构建重组表达载体pGEX-4T-2-MAGE-E1，并转化大肠杆菌进行表达。结果：1mmol／L．异丙基-β-D-硫代半乳糖苷(IPTG)诱导5h，MAGE-E1蛋白表达即达高峰，SDS-PAGE及凝胶密度扫描分析表明，表达出Mr约41000大小的蛋白，占菌体总蛋白的35％。结论：成功扩增、克隆MAGE-E1基因，并在大肠杆菌中得到稳定高效表达。

Cloning, sequencing and expression of human testicular carcinoma antigen MAGE-E1 gene

Objective To amplify and to clone the human MAGE-E1 gene and to express its recombinant protein in E.coli. Methods The cDNA encoding human MAGE-E1 gene was amplified by RT-PCR from human glioma cell line BT-325, then the MAGE-E1 gene was inserted into plasmid pGEM-T easy. After sequencing, the MAGE-E1 was cloned into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant expression vector pGEX-4T-2-MAGE-E1 and it was used to transform E.coli. Results The expressed product reached the highest level at 5 h after IPTG induction. SDS-PAGE and scanning analysis of gel density indicated that the expressed protein was about Mr 41 000 and account to 35% of the total bacterial protein. Conclusion The human testicular carcinoma antigen MAGE-E1 has been successfully cloned and efficiently expressed in E.coli.