以大肠埃希菌tolC蛋白为靶标的适配体的筛选及结构分析  

Screening and structure analysis of the aptamer target to Escherichia coli tolC protein

作　　者：陈伶利[1] 李杰[1] 张晓青[1] 宋岚[1] 钱纯[1] 葛金文[1] 

机构地区：[1]湖南中医药大学医学院,长沙410208

出　　处：《北京大学学报（医学版）》2014年第5期698-702,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81202451;81000026);湖南省自然科学基金(12JJ4029);高等学校博士学科点专项科研基金(20114323120002);中国博士后基金(2012M511731);湖南省教育厅科学研究项目重点项目(11A083);湖南省高校科技创新团队基金(15);湖南省重点学科基金(100601)资助~~

摘　　要：目的:筛选大肠埃希菌外排泵tolC蛋白的核酸适配体。方法:重组表达大肠埃希菌外排泵外膜蛋白tolC,利用指数富集的配体系统进化技术(stematic evolution of ligand by exponential enrichment,SELEX)从单链DNA文库中筛选出一组能够特异性与其结合的核酸适配体。利用FITC荧光素标记技术检测每轮单链DNA文库与靶蛋白的亲和力,直至亲和力的上升趋于饱和,将最后一轮筛选产物克隆测序,并用DNAman软件分析其二级结构。结果:经过12轮筛选,单链DNA文库与靶标蛋白的亲和力趋向稳定,将第12轮筛选产物克隆测序,对获得的23个适配体进行分析。一级结构分析显示23个适配体并无共同的保守序列,但分别有3对和2对适配体序列完全一致。二级结构预测分析表明,茎环结构为适配体主要的结构形式,提示其可能是适配体与tolC蛋白蛋白特异性结合的基础。根据二级结构特点可将23个适配体分为4个家族,其中20号适配体与靶标蛋白亲和力最高。结论:成功利用SELEX技术筛选获得了特异结合tolC的高亲和力的核酸适配体,为大肠埃希菌耐药干预及机制研究奠定了基础。Objective：To screen and characterize the aptamer of Escherichia coli outer member protein tolC. Methods： By using the recombinant E. coli outer member protein tolC for the screening target,oligonucleotides which were capable of specifically binding to the protein were screened from a random oligonucleotide library through the stematic evolution of ligand by exponential enrichment（ SELEX） technique. The binding capacity of ssDNA to the targeted protein from each round was detected by the FITC fluorescence labeling technique. The ssDNA from the last cycle was cloned and sequenced,and the second structure was further analyzed by the DNAMan program. Results： After 12 cycles of selection,40 clones were selected randomly and sequenced. Although a unique conserved sequence was not obtained among the 23 obtained aptamers by the primary structure analysis,three pairs of aptamers and two pairs of aptamers were found to be identical. Analysis of the secondary structure revealed that the stem-loop and bulge loop were the main motifs,indicating that they might play a key role in the binding of aptamers to the target protein. According to the characteristic of the second structure,23 aptamers were divided into four families,and aptamer 20 bore the greatest affinity. Conclusion： Aptamers against E. coli outer member protein tolC were successfully identified by the SELEX method. The results laid a foundation for the investigation of the interference to the drug resistance of E. coli and the underlying mechanisms.

关 键 词：适体 核苷酸 tolC蛋白 大肠杆菌 抗药性 

分 类 号：R965.1[医药卫生—药理学]