迁移侵袭抑制蛋白 MIIP 基因 K167E 位点突变体的构建和表达

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eDNA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B121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。

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

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 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.