应用噬菌体展示技术筛选HMGB1启动子结合蛋白  

Screening of binding proteins of HMGB1 promoter by phage display technique

作　　者：丁宁[1] 肖慧[2] 高巨 许立新[1] 佘守章[1] 

机构地区：[1]广州市第一人民医院麻醉科和麻醉学实验室,广东广州510180 [2]广州市第一人民医院门诊部,广东广州510180 [3]广州中医药大学第二附属医院麻醉科,广东广州510120

出　　处：《中国病理生理杂志》2010年第1期28-31,共4页Chinese Journal of Pathophysiology

基　　金：广州市医药卫生科技资助项目(No.2008-YB-012;No.2009-YB-021);广州市医药卫生科技重点资助项目(No.2008-ZDi-14);广东省科技计划资助项目(No.2007B31509007);广东省医学科研基金资助项目(No.A2009497)

摘　　要：目的:筛选高迁移率族蛋白B1(HMGB1)启动子结合蛋白。方法:应用噬菌体展示技术,以HMGB1启动子DNA作为固相筛选分子,对噬菌体展示环七肽库进行4轮"吸附-洗脱-扩增"生物筛选,从第4轮洗脱物中随机挑选20个单克隆噬菌体扩增后进行ELISA鉴定。对所筛选克隆进行DNA序列测定和生物信息学分析。结果:经过4轮筛选后,对随机选取的20个噬菌体克隆进行鉴定,其中11个可与HMGB1启动子结合。DNA测序后经过同源性搜索,确定了与HMGB1启动子具有结合作用的蛋白,包括激活蛋白-1(activator protein-1,AP-1)等6种已知功能蛋白和2种未知功能蛋白。结论:筛选到HMGB1启动子的结合蛋白,为研究HMGB1基因的转录调控机制奠定基础。AIM： To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS： HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme - linked immunosorbent assay （ELISA）. Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS ： Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein - 1 （ AP - 1 ） and two of which were uncharacterized ones. CONCLUSION： Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.

关 键 词：高迁移率族蛋白1 启动子 噬菌体展示 肽库 基因表达调控 

分 类 号：R378[医药卫生—病原生物学] R392.1[医药卫生—基础医学]