脓毒症单核巨噬细胞特异性结合肽的筛选  

Screening of Peptides Specifically Binding to Monocyte/Macrophage in Sepsis

作　　者：丁宁[1] 刘承武[1] 李志杰[1] 刘靖华[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室和广东省功能蛋白质组学重点实验室,广州510515

出　　处：《中国生物化学与分子生物学报》2007年第12期1045-1050,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973计划;No.2002CB513005);国家自然科学基金资助项目(No.30670828)资助;广东省科技计划项目(No.A1090202)~~

摘　　要：利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide,LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮"差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.By phage display technique, peptides bound specifically to lipopolysaccharide stimulated THP-1 cells were biopanned from a phage 7-mer cyclic peptide library. THP-1 cells stimulated by lipopolysaccharide were used as target cells and untreated THP-1 cells as absorbing cells. After four rounds of biopanning to a phage display heptapeptides library, individual phage clones were selected and identified by ELISA assay. Positive phage clones were characterized by DNA sequencing and bioinformatics analysis. The conserved motifs fused heptapeptides were identified with fluorescence microscopy. After four rounds of biopanning, all the clones were identified as positive clones. Multiple sequence alignment tool Clustal W and sequence similarity search program BlastP were used to analyze specific peptide sequences of nonredundant heptapeptides. Phage clones that display specific sequences obtained by bioinformatics bound specifically to LPS stimulated THP-1 cells with immunofluorescence analysis. Phage display technique provides an efficient selection system for screening specific targets locating on the cell surface. The bioactivities of the obtained peptides remain to be determined.

关 键 词：单核 巨噬细胞 脂多糖 噬菌体展示 肽库 脓毒症 

分 类 号：Q291[生物学—细胞生物学] Q34