睾丸肿瘤抗原MAGE-E1基因片段的克隆及其在大肠杆菌中的表达

Cloning of human testicular carcinoma antigen MAGE-E1 gene and its expression in E.coli

目的 :克隆及表达人胶质瘤特异性抗原MAGE E1基因片段。方法 :从人胶质瘤细胞系BT 32 5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 :用 1mmol/L异丙基 β D 硫代半乳糖苷 (IPTG)诱导 5h ,MAGE E1蛋白的表达即达高峰。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 :该基因片段的高效表达为进一步制备其抗体 ,以及制备肿瘤疫苗奠定了基础。

AIM: To clone and express the testicular carcinoma antigen MAGE E1 gene 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 which 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 M r 41 000 and account for 35% of the total bacterial protein. CONCLUSION: The High efficient expression of the MAGE E1 gene fragment lays the foundation for further preparing antibody against MAGE E1 protein and the tumor vaccine for glioma immunothrapy.