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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。
Objective: To clone and express human glioma antigen MAGE E1 gene fragment. METHODS: Total RNA was extracted from the human glioma cell line BT32 5 and the MAGE E1 gene fragment was amplified by RT PCR. The MAGE E1 gene fragment was inserted into the vector pGEM Teasy and sequenced by the automatic sequence analyzer. The MAGE E1 gene fragment was then cloned into the expression vector pGEX 4T 2 to construct the recombinant expression vector pGEX 4T 2 MAGE E1 and transformed into E. coli for expression. Results: The expression of MAGE E1 protein peaked at 1 h after induction with 1 mmol / L isopropyl β D thiogalactoside (IPTG). SDS PAGE and gel density scanning analysis showed that the expression of Mr about 4 10 0 0 protein, accounting for 35% of the total bacterial protein. Conclusion: The high expression of the gene fragment for further preparation of its antibody, and preparation of tumor vaccine laid the foundation.