NK4蛋白在大肠杆菌中的表达及其活性研究  

Expression of NK4 Protein in Escherichia coli and Its Biological Characterization Analysis

作　　者：罗建平[1] 滑世轩[1] 赵行[1] 岑东 吕建新[1] 涂植光[3] 裴仁治 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室,温州325027 [2]浙江省鄞州人民医院,宁波315040 [3]重庆医科大学医学检验系临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物工程杂志》2009年第10期33-37,共5页China Biotechnology

基　　金：浙江省医药卫生科学研究基金(2003B172;2007A175);宁波市医药卫生科学研究基金(2003079);宁波市科技计划(2007C10065)资助项目

摘　　要：NK4蛋白是近年来发现的肝细胞生长因子的最佳拮抗剂。为规模化生产NK4蛋白,将NK4基因插入载体pET-26b(+),构建重组原核表达载体pET-26b(+)-NK4,并转化大肠杆菌Rosseta(DE3)。转化菌经IPTG诱导后以包涵体形式大量表达重组蛋白,占菌体总蛋白的42%。包涵体用盐酸胍溶解后经Ni-NTA树脂亲和层析纯化,蛋白纯度约为95%,经Western blot证实为NK4蛋白。纯化的重组蛋白行稀释复性后可抑制Hela细胞的贴壁、迁徙,并诱导其凋亡,证实制备的NK4蛋白具有生物活性。NK4蛋白的成功制备将有助于NK4相关功能的深入研究。NK4 protein is considered as the best antagonist of hepatocyte growth factor in recent years.In order to produce and study the biological characteristics of NK4 protein,the complete coding sequences of NK4 gene were cloned into vector pET-26b（＋） to construct recombinant prokaryotic expression vector pET-26b（＋）-NK4.After induction with IPTG,the strain of E.coli Rosseta（DE3） transformed successfully with the recombinant expression vector expressed recombinant protein existed in format of inclusion body strongly.The inclusion body,42% of the total protein in E.coli,was dissolved by guanidine hydrochloride and purified by Ni-NTA column.The purified protein,about 95% purity,was confirmed by Western blot as NK4 protein.These results showed that recombinant protein could inhibit adherence,migration and induce apoptosis of Hela cell,which suggested that NK4 protein acquired retained its biological activity.The findings will be helpful to further study of NK4 function associated.

关 键 词：NK4蛋白 重组 表达 纯化 生物活性 

分 类 号：Q819[生物学—生物工程]