NK4蛋白在大肠杆菌中的表达及其纯化与活性研究  

Expression of NK4 protein in E. coli and its purification and biological characterization analysis

作　　者：岑东 罗建平[1] 赵行[2] 滑世轩[2] 吕建新[2] 涂植光[3] 裴仁治 

机构地区：[1]浙江省鄞州人民医院医学检验中心 [2]温州医学院浙江省医学遗传学重点实验室 [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

出　　处：《浙江检验医学》2010年第1期3-7,共5页Zhejiang Journal of Laboratory Medicine

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

摘　　要：目的通过构建NK4基因的重组原核表达载体,规模化制备NK4蛋白。方法将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E.coli Rosseta(DE3)。IPTG诱导转化菌表达NK4蛋白,然后采用Ni-NTA树脂亲和层析进行纯化。观察复性后NK4蛋白对Hela细胞的生物学性状的影响,评价制备的NK4蛋白的生物活性。结果NK4基因重组原核表达载体pET-26b(+)-NK4获成功构建。转化pET-26b(+)-NK4的E.coli Rosseta(DE3)以包涵体形式大量表达目的蛋白,占菌体总蛋白的42%。经Ni-NTA树脂亲和层析纯化后NK4蛋白纯度约为95%,并经Western blot证实。NK4蛋白行稀释复性后可抑制Hela细胞的贴壁、迁徙并促进其凋亡。结论成功构建NK4基因的重组原核表达载体pET-26b(+)-NK4,并可在E.coli Rosseta(DE3)表达。制备的NK4保留了其生物学活性。Objective To construct NK4 gene recombinant prokaryotic expression vector for the sake of NK4 protein scale production.Methods NK4 gene amplified by PCR was cloned into vector pET-26b(+)to construct its recombinant prokaryotic expression vector pET-26b(+)-NK4.When target protein was expressed in strain of E.coli Rosseta(DE3)transformed with the recombinant vector pET-26b(+)-NK4 by IPTG induction,it was dissolved by guanidine hydrochloride and then purified by Ni-NTA affinity chromatography.Biological characterization of renatured NK4 protein was estimated when NK4 protein was added into Hela cell culture to investigate its influence in Hela cell’s biological characters.Results NK4 gene recombinant prokaryotic expression vector pET-26b(+)-NK4 was constructed successfully.NK4 protein existed as inclusion body was strongly expressed in the strain of E.coli Rosseta(DE3)transformed with the recombinant vector pET-26b(+)-NK4,which was accounted about 42% of total thalline protein.It showed that purity of NK4 protein purified by Ni-NTA affinity chromatography and confirmed by Western blot was about 95%.Having been renatured,NK4 protein could inhibit Hela cell adherence,migration and induce its apoptosis.Conclusion NK4 gene recombinant prokaryotic expression vector pET-26b(+)-NK4 was successfully constructed and could express in transformed E.coli Rosseta(DE3).NK4 protein obtained retained its biological characterization.

关 键 词：NK4蛋白 表达 纯化 生物活性 

分 类 号：Q78[生物学—分子生物学]