突变人GM-CSF基因在大肠杆菌中的表达及纯化  

The expression and purification of mutant hGM-CSF gene in E.coli

作　　者：马莉[1] 白泉[1] 王骊丽[1] 耿信笃[1] 

机构地区：[1]西北大学现代分离科学研究所/现代分离科学陕西省重点实验室,陕西西安710069

出　　处：《西北大学学报（自然科学版）》2006年第1期63-67,共5页Journal of Northwest University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(20175016)

摘　　要：目的构建重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的高效表达质粒,将其表达产物用于分离纯化的研究。方法用人外周血单核细胞获得总RNA作为模板和修饰的5′端密码子的引物经RT-PCR反应扩增到人GM-CSF基因,插入温控表达载体pDH构建了突变的hGM-CSF表达质粒,并将在E.coli中获得表达的突变rhGM-CSF包含体8 mol/L脲提取液进一步用离子交换色谱柱分离纯化。结果rhGM-CSF在大肠杆菌表达占菌体总蛋白量的22%,用弱阴离子交换色谱对rhGM-CSF的工程菌表达产物8 mol/L脲提取液直接经35 m in分离纯化,所得产物纯度可达95%,质量回收率可达到60%。结论这表明用离子交换色谱可进行rhGM-CSF的复性与同时纯化。Aim To construct recombination of human granulocyte-macrophage colony stimulating factor （rhGM- CSF） with DNA recombination technologies and purification of weak anion ion exchange chromatography （WAX）. Methods The total RNA was obtained from monoc＇ytes of human peripheral blood as a template. The mutant hGM-CSF cDNA gene was amplified by RT-PCR method with the 5＇ terminal modified primer. Results The cDNA encoding mutant hGM-CSF was sequenced and inserted into the expression vector （pDH）. The mutant rhGM-CSF expressed in E. coli was purified with WAX. Conclusion The expression level of rhGM-CSF in E. coli DH5α was about 22%. By one step with 35 min, the extraction of rhGM-CSF with 8.0 mol/L urea solution was separated and purified with WAX. It was found that the purity and the mass recovery of tumant rhGM-CSF can be obtained to be 60% and 95 %, respectively. The result indicates that WAX can be used to investigate the renaturation with simultaneous purification of rhGM-SCF.

关 键 词：重组人粒细胞-巨噬细胞集落刺激因子 表达 大肠杆菌 离子交换色谱 分离纯化 

分 类 号：R318[医药卫生—生物医学工程]