| SELEX技术筛选毒死蜱单链DNA适体 |
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| 引用本文： | 雷兆静,张存政,刘媛,王丽,胡秋辉,刘贤进.SELEX技术筛选毒死蜱单链DNA适体[J].江苏农业学报,2012(1):198-203. |
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| 作者姓名： | 雷兆静 张存政 刘媛 王丽 胡秋辉 刘贤进 |
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| 作者单位： | 南京农业大学食品科技学院;江苏省农业科学院食品质量安全检测研究所农业部食品安全监控重点开放实验室 |
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| 基金项目： | 江苏省农业科技自主创新基金项目[CX(10)236] |
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| 摘 要： | 试验旨在利用SELEX技术体外筛选毒死蜱的特异性适体。体外合成全长为91 nt的ssDNA文库,以链亲和素修饰的凝胶为载体、毒死蜱为靶分子进行SELEX(配体指数增强系统进化技术)筛选。利用荧光标记法测定适体的筛选效率、亲和力和特异性,通过MFOLD分析软件对亲和力较高的适体进行二级结构预测和结合位点分析。结果表明,经过15轮筛选后,DNA文库的筛选效率达到44.00%;最终获得9条ssDNA适体,其中适体N23对毒死蜱具有最高的亲和力,其结合活性显著高于N23与水胺硫磷、丙溴磷、氧化乐果的结合活性;二级结构表明茎环结构可能是毒死蜱与适体相互作用的结构基础。
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| 关 键 词： | 适体 SELEX 荧光 毒死蜱 |
Selection of chlorpyrifos-binding ssDNA aptamer by SELEX |
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| LEI Zhao-jing,ZHANG Cun-zheng,LIU Yuan,WANG Li,HU Qiu-hui,LIU Xian-jin.Selection of chlorpyrifos-binding ssDNA aptamer by SELEX[J].Jiangsu Journal of Agricultural Sciences,2012(1):198-203. |
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| Authors: | LEI Zhao-jing ZHANG Cun-zheng LIU Yuan WANG Li HU Qiu-hui LIU Xian-jin |
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| Institution: | 1.College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;2.Institute of Food Quality Safety and Detection,Key Laboratory of Food Safety Monitoring and Management,Ministry of Agriculture,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China) |
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| Abstract: | This experiment aimed to select specific aptamer of chlorpyrifos by SELEX.With streptavidin agarose resinsn as carrier and chlorpyrifos as target,a synthetic DNA library with total length of 91 nt was subjected to selection using SELEX(Systematic evolution of ligands by exponential enrichment) technology.By fluorescent label technique,the selection efficiency,affinity and specificity of selected aptamer were investigated.The secondary structures of aptamers with higher affinities and their corresponding binding sites were predicted and analyzed by MFOLD software.The results showed that after 15 rounds in vitro selection,the efficiency of selection could reach 44.00%.Nine aptamers were successfully selected for their high abilities to bind chlorpyrifos,of which,aptamer N23 was the best for binding chlorpyrifos.The stem-loop of aptamer might work as the main motif in the interaction between aptamer and chlorpyrifos. |
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| Keywords: | aptamer SELEX fluorescence chlorpyrifos |
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