作　　者： ; ; ; ; ; (文露婷）;

机构地区： 西南大学动物科技学院动物医学系

出　　处： 《中国兽医学报》 2016年第6期1008-1014,共7页

摘　　要： 核酸适配体作为一种优于单克隆抗体的新型识别分子,具有更高的分辨率,能识别配体间一个基团的细微差别,且所需的结合活性位点更小。本研究利用指数级富集配体进化技术(SELEX)筛选特异性结合于奶牛结合珠蛋白(HP)的DNA适配体,并对筛取的适配体进行特性分析。体外构建全长80bp中间含40bp随机序列的单链DNA(ssDNA)文库,通过对循环次数、退火温度及引物比例的优化,建立了适合的筛选体系;以超滤离心管为介质,HP蛋白为靶标,对随机ssDNA文库进行反复的SELEX筛选,从单链DNA文库中筛选能特异性结合HP蛋白的核酸适配体;利用DNAMAN和RNAstructure软件对获得核酸适配体进行一级结构和二级结构分析。结果表明,经过体外8轮筛选,随机ssDNA文库与HP蛋白的结合率由1.86%上升到31.35%,特异性核酸达到最大富集;经克隆和测序,获得了能与HP蛋白特异性结合的5个家族的DNA适配体,同源性均达70%以上,RNAstructure分析其二级结构主要以茎环结构为主,表明ssDNA形成不同的茎环结构可能是适配体与HP蛋白特异性作用的结构基础。初步建立HP蛋白核酸适配体库,筛选到与HP蛋白特异性结合的核酸适配体,为制备以HP为检测靶标的奶牛隐性乳腺炎检测试剂盒奠定基础。 Aptamer as a newly recognized molecular has many advantages compared with monoclonal antibody,it has higher resolution and could recognize tiny difference of a group between the ligands and needs smaller active site to bind.This experiment was designed to screen the specific binding aptamers against bovine haptoglobin（HP）using the technology of system evolution of ligands by exponential enrichment（SELEX）and the specificity of selected aptamers were analyzed.An 80 nt primary ssDNA library containing 40 bp random oligonucleotides was synthesized in vitro.Through the optimization of critical PCR and asymmetric PCR parameters including cycles,annealing temperature and ratio of primers,a suitable screening system was established.The specific binding aptamers against HP protein were obtained from random ssDNA library by iterative SELEX procedures performed with the target protein of haptoglobin and the mediator of amicon ultra-0.5 mL centrifugal filters.Primary and secondary structure of selected aptamers were analyzed by DNAMAN and RNAstructure software,respectively.The results showed that the binding rate of random ssDNA to HP protein increased from 1.86%to 31.35%after 8rounds selection,indicating that specific aptamers were enriched progressively.Five specific bindingaptamers against HP protein with at least about 70% homology in different family were obtained after amplifying,cloning and sequencing.The result of ssDNA aptamers secondary structure predication by RNAstructure software showed that the spatial structure of selected ssDNA aptamers were mainly characterized by stem-loops.The stem-loops might be the specific binding structural basis between the aptamer and HP protein.Aptamer library of HP protein was initially established,and nucleic acid aptamers with high binding affinity and specificity against HP protein were obtained,which lay a foundation for the future application of these aptamers in specific detection of subclinical bovine mastitis by the detected target marker of HP protein.

关 键 词： 奶牛乳腺炎 结合珠蛋白 适配体

领　　域： [农业科学] [农业科学] [农业科学] [农业科学] [农业科学] [农业科学]