摘要
利用SELEX技术从一个固定的随机ssDNA库中同时筛选甲拌磷、丙溴磷、水胺硫磷、氧化乐果4种有机磷农药的DNA适体。随着筛选轮数的增加,4种有机磷农药从固定的随机库中洗脱的ssDNA逐渐富集,经过12轮的筛选,筛选效率达到40.8%。对第12轮洗脱的ssDNA进行克隆、测序,挑选的15个阳性克隆均测序成功,由于部分克隆具有完全一致的序列,最终筛选到5条ssDNA序列。一级结构序列分析表明5条ssDNA具有一定的同源性,二级结构预测表明5条ssDNA的二级结构主要以茎环结构为主,表明茎环结构可能是适体结合4种有机磷农药的基础。
DNA aptamers against four organophosphorus pesticides （phorate, profenofos, isocarbophos and omethoate ）were simultaneously isolated from an immobilized random ssDNA library by systematic evolution of ligands by exponential enrichment （SELEX） technique. During each round of selection, the ssDNAs binding to the organophosphorus pesticides were eluted from the im- mobilized ssDNA library, and enriched as the selection round progressed. At the twelfth selection round, the selection efficiency was 40.8% and peaked. The eluted ssDNAs of the twelfth selection were amplified and cloned into a pUC-T simple vector. Fifteen positive colonies with plasmids containing inserted ssDNA were sequenced. Top 5 ssDNAs with the highest homogeneity in sequence were selected for further characterization. First structures and second structures of 5 ssDNAs were analyzed, which showed that they were homologous, and typically assembled by loop motifs. The loop motifs might be the binding basis between the aptamer and four organophosphorus pesticides.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2013年第6期117-122,共6页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31201369)
江苏省农业自主创新项目[CX(10)236]
关键词
有机磷农药
DNA适体
指数富集的配体系统进化(SELEX)
筛选
结构分析
organophosphorus pesticide
DNA aptamer
systematic evolution of ligands by exponential enrichment （SELEX）
selection
structure analysis
