首页 | 本学科首页   官方微博 | 高级检索  
检索        

NK4蛋白在大肠埃希菌中的表达、纯化、复性及活性测定
引用本文:常冰梅,刘晓军,李美宁,张悦红,程牛亮,牛勃.NK4蛋白在大肠埃希菌中的表达、纯化、复性及活性测定[J].复旦学报(医学版),2011,38(1):60-65.
作者姓名:常冰梅  刘晓军  李美宁  张悦红  程牛亮  牛勃
作者单位:山西医科大学生物化学与分子生物学教研室-细胞生理学省部共建教育部重点实验室,太原030001;中国医学科学院基础医学研究所北京100005;首都儿科研究所,北京100020
基金项目:山西省科技攻关项目(20080311059-6); 山西医科大学科技创新项目(01200713)
摘    要:目的 在大肠埃希菌(E. coli)中高表达NK4蛋白,经鉴定、纯化、复性后测定其生物学活性.方法 采用重组DNA技术对已有质粒pGEX-4T-1-NK4和pBV220-HGFα进行改造,构建质粒pBV220-NK4,并使其转化E. coli BL21(DE3),温控诱导表达目的 蛋白NK4.蛋白鉴定采用SDS-PAG...

关 键 词:NK4  原核表达  纯化  活性测定
收稿时间:2010-7-16

Expression, purification, renaturation and activity assay of NK4 in Escherichia coli
CHANG Bing-mei,LIU Xiao-jun,LI Mei-ning,ZHANG Yue-hong,CHENG Niu-liang,NIU Bo.Expression, purification, renaturation and activity assay of NK4 in Escherichia coli[J].Fudan University Journal of Medical Sciences,2011,38(1):60-65.
Authors:CHANG Bing-mei  LIU Xiao-jun  LI Mei-ning  ZHANG Yue-hong  CHENG Niu-liang  NIU Bo
Institution:Key Laboratory of Cellular Physiology, Ministry of Education-Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China; Capital Institute of Paediatrics, Beijing 100020, China
Abstract:Objective To construct a recombinant plasmid expressing NK4 gene in Escherichia coli (E. coli) and to obtain purified NK4 protein with bioactivity. Methods The plasmid pBV220-NK4 was constructed by recombinant technology with utilization of the plasmid pBV220-HGFα and pGEX-4T-1-NK4. The plasmid pBV220-NK4 was identified by the restriction analysis and DNA sequencing. Then recombinant plasmid was transformed into E. coli and the recombinant protein was expressed and detected by SDS-PAGE and Western blot. The expressed protein existing in the form of inclusion body was purified by gel filtration chromatography, and then was detected for the activity by MTT method after renaturation. The expression of the proliferating cell nuclear antigen (PCNA) in human umbilical vein endothelial cells (HUVEC) ECV304 incubated with NK4 was measured by immunohistochemical strain. Results SDS-PAGE showed a specific protein band with relative molecular weight of 49 000. The expressed product existed in the form of insoluble inclusion body and contained about 30% of total somatic protein. After renaturation, the expressed NK4 showed an inhibitory action on ECV304 proliferation by MTT and the ratio of inhibition was 30% (P<0.01). The expressed NK4 also obviously depressed the expression of PCNA. Conclusions NK4 protein was successfully expresseed in E. coli, which layed the foundation for the study on the structure and function as well as pilot production of NK4.
Keywords:NK4  prokaryotic expression  purification  activity assay  
本文献已被 CNKI 万方数据 等数据库收录!
点击此处可从《复旦学报(医学版)》浏览原始摘要信息
点击此处可从《复旦学报(医学版)》下载免费的PDF全文
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号