以大肠埃希菌tolC蛋白为靶标的适配体的筛选及结构分析

发布时间：2018-01-07 18:42

本文关键词：以大肠埃希菌tolC蛋白为靶标的适配体的筛选及结构分析　出处：《北京大学学报(医学版)》2014年05期 　论文类型：期刊论文

【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。
[Abstract]:Objective: to screen the nucleic acid aptamer of Escherichia coli efflux pump tolC protein. Methods: recombinant expression of Escherichia coli efflux pump outer membrane protein tolC. Ligand phylogenetic techniques with exponential enrichment (. Stematic evolution of ligand by exponential enrichment. Selex) a group of nucleic acid aptamers that can specifically bind to single strand DNA library were screened. The affinity of each round of single stranded DNA library to target protein was detected by FITC fluorescein labeling technique. Until the affinity increased to saturation, the final screening product was cloned and sequenced, and its secondary structure was analyzed by DNAman software. Results: after 12 rounds of screening. The affinity of the single strand DNA library to the target protein tended to be stable and the 12th screening product was cloned and sequenced. The analysis of the 23 aptamers obtained showed that the 23 aptamers had no conserved sequence, but 3 pairs and 2 pairs of aptamers were identical respectively. The secondary structure prediction analysis showed that there was no conserved sequence in the 23 aptamers. Stem ring structure is the main structural form of aptamer, suggesting that it may be the basis of specific binding between aptamer and tolC protein. According to the secondary structure, 23 aptamers can be divided into 4 families. Among them, the aptamer 20 has the highest affinity to the target protein. Conclusion: high affinity aptamers with specific binding to tolC were successfully screened by SELEX technique. It lays a foundation for drug resistance intervention and mechanism study of Escherichia coli.
【作者单位】：湖南中医药大学医学院;
【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~~
【分类号】：R3416
【正文快照】： Province(15),and the Construct Program of the Key Discipline In Hunan Province(100601).△Corresponding author’s e-mail,cmgjw@tom.com大肠埃希菌多重耐药主要与主动外排系统有关,其外排基因占所有转运蛋白基因的6%~18%[1-3],因此抑制大肠埃希菌的主动外排泵对解决其

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