全细菌SELEX技术筛选鸟分枝杆菌的适配子  被引量:4

Aptamers from whole-bacterium SELEX targeting Mycobacterium avium

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作  者:王培培[1] 刘焰[1] 罗凤玲[1] 章晓联[1] 

机构地区:[1]武汉大学基础医学院免疫学系,病毒学国家重点实验室,湖北省过敏及免疫相关疾病重点实验室,湖北武汉430071

出  处:《中国病原生物学杂志》2015年第2期113-117,179,共6页Journal of Pathogen Biology

基  金:国家重大传染病专项(No.2012ZX10003002-015);国家自然科学基金项目(No.31270176);湖北省自然科学基金项目(No.2014CFB719);湖北省医学领军人才项目(No.523-276003);教育部博士点基金(No.20100141120056)

摘  要:目的应用指数级富集配体系统进化技术(system evolution of ligands by exponential enrichment,SELEX)筛选与鸟分枝杆菌(Mycobacterium avium)特异性结合的ssDNA适配子,建立酶联寡聚核苷酸吸附试验(enzyme-linked oligonucleotide assay,ELONA)检测鸟分枝杆菌的方法。方法设计并合成随机序列ssDNA文库,以鸟分枝杆菌为靶标,利用SELEX技术筛选特异性的ssDNA适配子,将亲和力最高的适配子进行克隆、测序;用DNAMAN软件分析适配子的二级结构,并检测其特异性。结果单适配子N1与鸟分枝杆菌结合的Kd为(246±21.87)nmol/L,即亲和力最高,而对结核分枝杆菌等其他分枝杆菌亲和力较低。结论应用SELEX筛选的鸟分枝杆菌ssDNA适配子N1具有较高亲和力和特异性,可作为鸟分枝杆菌检测及艾滋病合并鸟分枝杆菌感染的特异性诊断制剂。Objectives To use systematic evolution of ligands by exponential enrichment(SELEX)to screen for ssDNA aptamers specifically targeting Mycobacterium avium and to establish a new enzyme-linked oligonucleotide assay(ELONA)to detect M.avium. Methods A random ssDNA library was designed and synthesized to screen for ssDNA aptamers specifically targeting M.avium.High-affinity aptamers were cloned and sequenced.The secondary structure of aptamers was analyzed using DNAMAN software,and the specificity of aptamers was determined using ELONA. Results Aptamer N1 had the highest affinity for and specificity to M.avium.The Kd for aptamer N1 and M.avium was246±21.87nmol/L.Aptamer N1 specifically bound with M.avium but not with M.tuberculosis and other mycobacteria. Conclusion SELEX yielded aptamer N1,which had a high affinity for and specificity to M.avium.This aptamer could serve as a reagent for diagnosis of M.aviumand patients coinfected with AIDS and M.avium.

关 键 词:鸟分枝杆菌 SELEX 适配子 

分 类 号:R378.91[医药卫生—病原生物学]

 

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