脆性X智力低下蛋白在大肠杆菌中的表达及纯化  

Expression and purification of human fragile X mental retardation protein in Escherichia coli

作　　者：李芳芳[1,2] 郭永[1] 崔芳芳[1,3] 丁宁[1] 张嵘[2] 邹检平[1] 

机构地区：[1]国家人口计生委科学技术研究所,北京100081 [2]沈阳药科大学生命科学与生物制药学院 [3]河南大学药学院

出　　处：《中国计划生育学杂志》2012年第6期410-414,共5页Chinese Journal of Family Planning

摘　　要：目的:在大肠杆菌中对脆性X智力低下蛋白(FMRP)进行重组表达,获得高产量及高纯度的可溶性重组蛋白。方法:以人脑cDNA文库为模板,PCR扩增FMR1基因后将其克隆至融合表达载体pGEX-6P-1中,重组载体转化进入大肠杆菌BL21(DE3)pLysS,并用IPTG进行诱导表达。应用谷胱甘肽-琼脂糖凝胶4B进行亲和层析纯化GST-FMRP融合蛋白,并以SDS-PAGE电泳和Westernblot对融合蛋白的表达进行检测。结果:亲和纯化后的融合蛋白经FMRP及GST单抗分别进行Westernblot鉴定为GST-FMRP融合蛋白。结论:大肠杆菌中FMRPISO10的重组表达产量高,纯化得到可溶性重组蛋白,为FMRP抗体的制备及FXS检测试剂盒的开发奠定基础。Objective ： To obtain sufficient and highly purified soluble recombinant fragile X mental retardation protein （ FM-RP） in Escheriehia coli. Methods： With human brain eDNA library as template, FMR1 gene fragments were obtained by PCR and inserted into the pGEX - 6P - 1 plasmid. The recombinant vector was transformed into host E. coli BL21 （ DE3 ） pLysS and the protein expression was induced by IPTG. The fusion recombinant protein was purified by Glutathione sepharose 4B af-finity chromatography and detected with SDS - PAGE and Western blot. Results ： The purified recombinant protein was con-firmed to be GST - FMRP fusion protein by Western blot with anti - FMRP and anti - GST monoclonal antibodies. Conclu- sion： The recombinant FMRP ISO10 can be successfully expressed in Escherichia coll. The soluble FMRP ISO10 with a high - yield and purified expression level lays a foundation for antibody production and the development of FXS diagnosis kit.

关 键 词：脆性X综合征 FMRP重组表达 蛋白纯化 

分 类 号：R749.94[医药卫生—神经病学与精神病学]