甲基化CpG结合域重组蛋白的表达与鉴定  

Expression and identification of recombinant MBD proteins

作　　者：徐建[1,2] 潘世扬[1,2] 许雨乔[2] 孙瑞红[2] 黄蕾[2] 彭珊珊[2] 

机构地区：[1]南京医科大学第一附属医院检验医学部,国家临床检验重点专科,江苏南京210029 [2]南京医科大学医学检验系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2012年第11期1511-1515,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(30972821,30901262);江苏省自然科学基金(BK2009440);江苏省实验诊断学重点实验室基金(XK201114);江苏省高等学校大学生实践创新训练计划(ky102j201205)

摘　　要：目的:在大肠杆菌中表达甲基化CpG结合域(methyl-CpG-binding domain,MBD)重组蛋白。方法:对人甲基化CpG结合蛋白2(methyl-CpG-binding protein 2,Mecp2)的MBD区行密码子优化,将人工合成的DNA克隆至原核表达载体pGS21a,在大肠杆菌E.coli Rosetta(DE3)中诱导表达,通过SDS-PAGE和Western blot鉴定蛋白表达;镍亲和层析柱纯化重组蛋白。表面等离子共振分析重组MBD蛋白与甲基化DNA的结合能力。结果:酶切和核酸测序证实,成功构建了含密码子优化的MBD基因的原核表达载体。SDS-PAGE和Western blot结果显示,MBD重组蛋白在大肠杆菌中得到表达。亲和层析法纯化后获得了相对分子量为38 000的MBD重组蛋白。SPR分析显示MBD重组蛋白能特异结合甲基化DNA。结论:成功构建含密码子优化的MBD基因的原核表达载体,MBD重组蛋白能在大肠杆菌中表达。Objective：To express methyl-CpG-binding domain （MBD） recombinant proteins in E. coll. Methods：DNA encoding MBD of human methyl-CpG-binding protein 2 （Mecp2） was optimized for E. coli preferred codons. The codon-optimized MBD coding sequence was synthesized,and then was subcloned into pGS21a to construct recombinant expression plasmid. Recombinant protein expression was induced in E. coli Rosseta-gami （DE3）. Recombinant proteins were detected by SDS-PAGE and Western blot,and then purified using Nickel-affinity chromatography columns. The interaction between recombinant MBD proteins and methylated DNA was analyzed by surface plasmon resonance （SPR）. Results：Enzyme digestion and DNA sequencing demonstrated the prokaryotic expression vector carrying codon-optimized MBD gene was successfully constructed. SDS-PAGE and Western blot results indicated that recombinant MBD proteins were expressed in E. coll. Finally,recombinant MBD proteins with the relative molecular weight of 38 000 were purified by affinity chromatography. SPR analysis demonstrated that the recombinant MBD proteins could bind methylated DNA specially. Conclusion：The prokaryotic expression vector carrying codon-optimized MBD gene was successfully constructed,and recombinant MBD proteins were expressed in E. coli scucessfully.

关 键 词：甲基化CpG结合域 甲基化CpG结合蛋白2 原核表达 

分 类 号：Q786[生物学—分子生物学]