贵州医科大学学报

2008, (04) 340-343

[打印本页] [关闭]
本期目录(Current Issue) | 过刊浏览(Past Issue) | 高级检索(Advanced Search)

串联重复核定位信号的绿色荧光蛋白融合载体的构建与原核表达
Construction and Prokaryotic Expression of Green Fluorescent Protein Fusion Vector of Tandem Nuclear Localization Signals

邓鹏;陈腾祥;龚小卫;
DENG Peng,CHEN Tengxiang,GONG Xiaowei (Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province,Southern Medical University,Guangzhou 510515,Guangdong,China)

摘要(Abstract):

目的:构建绿色荧光蛋白-串联重复核定位信号融合蛋白的表达载体(His-EGFP-3xNLS),并在原核细胞中进行表达与纯化。方法:采用PCR方法从原先克隆在pEGFP-C1载体中的3xNLS与EGFP编码序列扩增出来,使用常规酶切和连接方法将其重组至pET-14b载体中,对阳性克隆进行酶切、PCR和测序鉴定。转化BL21(DE3)大肠杆菌株,用IPTG诱导融合蛋白表达,并利用Ni-NTA亲和层析纯化该融合蛋白。结果:构建的His-EGFP-3xNLS融合蛋白表达载体是正确的,该载体可在大肠杆菌内高效表达,用Ni-NTA纯化获得了相对分子质量约36 kD的融合蛋白。结论:成功构建了His-EGFP-3xNLS融合蛋白表达载体,并在原核细胞中获得了高效表达和纯化,为进一步研究NLS介导信号分子入核提供了实验材料。
Objective: To construct the expression vector of His-EGFP-3xNLS fusion protein and obtain its expression and purification in E.coli.Methods: 3xNLS and EGFP sequence was amplified by PCR from pEGFP-C1-3xNLS vector and cloned into pET-14b vector following the routine procedures.After identification by enzyme digestion,PCR and sequencing,the positive clones were transformed into BL21(DE3) competent cells,and the expression of His-EGFP-3xNLS fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography.Results: The constructed His-EGFP-3xNLS fusion protein vector highly expressed in E.coli.With Ni-NTA affinity chromatography,a purified His fusion protein with relative molecular mass of approximately 36 kD was obtained.Conclusion: The expression vector of His-EGFP-3xNLS fusion protein is constructed,expressed and purified under non-denaturing conditions,which may significantly facilitate future study of the mechanism by which NLS mediate the nuclear translocation of certain signal molecules.

关键词(KeyWords): 核定位信号;载体蛋白质类;基因表达;蛋白纯化
nuclear localization signal;carrier proteins;gene expression;protein purification

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(No.30700291)

作者(Author): 邓鹏;陈腾祥;龚小卫;
DENG Peng,CHEN Tengxiang,GONG Xiaowei (Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province,Southern Medical University,Guangzhou 510515,Guangdong,China)

Email:

DOI:

参考文献(References):

文章评论(Comment):

序号(No.) 时间(Time) 反馈人(User) 邮箱(Email) 标题(Title) 内容(Content)
反馈人(User) 邮箱地址(Email)
反馈标题(Title)
反馈内容(Content)
扩展功能
本文信息
服务与反馈
本文关键词相关文章
本文作者相关文章
中国知网
分享