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  

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

作　　者：王莉[1] 李青[1] 郭爱林[1] 郭斌[2] 张丰[1] 樊代明[3] 

机构地区：[1]第四军医大学西京医院病理科,西安710033 [2]第四军医大学研究生院 [3]第四军医大学西京医院消化内科

出　　处：《肿瘤》2003年第5期380-382,共3页Tumor

基　　金：国家自然科学基金资助项目 (编号 .3 0 10 0 2 18);高等学校骨干教师资助计划 (编号 .2 0 0 0 65 66);留学归国人员科研启动基金资助项目 (编号 1999 747)

摘　　要：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 32 5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 Teasy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 4 10 0 0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。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.

关 键 词：人睾丸肿瘤抗原 MAGE-E1基因 克隆 测序 原核系统 表达 

分 类 号：R730.231[医药卫生—肿瘤]