迁移侵袭抑制蛋白MIIP基因K167E位点突变体的构建和表达  

Construction and prokaryotic expression of migration and invasion inhibitor protein plasmid with K167E point mutation

作　　者：高玉婧[1] 马晓东[2] 芦晓红[3] 姚青[1] 裴秀英[1] 杨怡[1] 李建宁[1] 张茜[1] 孙玉宁[1] 

机构地区：[1]宁夏医科大学基础医学院生物化学与分子生物学系,宁夏银川750004 [2]宁夏人民医院,宁夏银川750021 [3]宁夏医科大学基础医学院,宁夏银川750004

出　　处：《宁夏医学杂志》2014年第1期7-10,I0001,共5页Ningxia Medical Journal

基　　金：国家自然科学基金资助项目(81101601);宁夏自然科学基金资助项目(NZ11125)

摘　　要：目的构建人迁移侵袭抑制蛋白(MIIP)蛋白K167E突变体的原核表达载体pGEX-4T-1-MIIP(K167E)并进行表达和纯化,为MIIP的后续功能研究提供有效工具。方法以pGEX-4T-1-MIIP重组质粒为模板,PCR扩增得到人MIIP基因(K167E)突变体的cDNA全长,将其克隆至原核表达载体pGEX-4T-1,构建获得pGEX-4T-1-MIIP(K167E)突变体,经酶切鉴定及测序验证完全正确后,将其转化至大肠杆菌BL21细胞中诱导表达,纯化获得GST-MIIP(K167E)融合蛋白。结果酶切鉴定和测序结果显示,pGEX-4T-1-MIIP(K167E)突变体表达载体构建成功。经诱导表达及纯化后,成功获得的GST-MIIP(K167E)融合蛋白。结论成功构建了pGEX-4T-1-MIIP(K167E)突变体表达载体,并获得了GST-MIIP(K167E)融合蛋白。Objective To construct the migration and invasion inhibitor protein （MIIP） plasmid with K167E point mutation for protein expression and purification, providing a useful tool for the study of MIIP~ role in cancer. Methods Whole MIIP （ K167E ） mu- tant coding sequence was obtained by PCR using pGEX - 4T - 1 - MIIP as the template, and then, it was cloned into pGEX - 4T - 1 plasmid to generate novel recombinant plasmid pGEX - 4T - 1 - MIIP （ KI67E ）. After identified by restriction enzyme digestion and se- quencing, the recombinant plasmid was transformed into E. coli BL21 to express, the target protein GST- MIIP （K167E） was obtained after purification. Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of pGEX - 4T - 1 - MIIP （K167E） was successfully constructed. After induced expression and purification, GST - MIIP （K167E） fusion protein was suc- cessfully obtained. Conclusion Prokaryotic expression recombinant plasmids of MIIP gene K167E point mutation is successfully con- structed. The fusion protein of GST- MIIP （KI67E） has been obtained.Objective To construct the migration and invasion inhibitor protein （MIIP） plasmid with K167E point mutation for protein expression and purification, providing a useful tool for the study of MIIP~ role in cancer. Methods Whole MIIP （ K167E ） mu- tant coding sequence was obtained by PCR using pGEX - 4T - 1 - MIIP as the template, and then, it was cloned into pGEX - 4T - 1 plasmid to generate novel recombinant plasmid pGEX - 4T - 1 - MIIP （ KI67E ）. After identified by restriction enzyme digestion and se- quencing, the recombinant plasmid was transformed into E. coli BL21 to express, the target protein GST- MIIP （K167E） was obtained after purification. Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of pGEX - 4T - 1 - MIIP （K167E） was successfully constructed. After induced expression and purification, GST - MIIP （K167E） fusion protein

关 键 词：pGEX-4T-1-MIIP GST—MIIP 突变 原核表达 融合蛋白 

分 类 号：R730.2[医药卫生—肿瘤]