中文摘要：

设计了一个长度为20个核苷酸的分子信标,建立了有机磷农药和分子信标竞争结合适配体鉴定其活性的方法,对前期筛选的两条适配体进行了活性位点分析和改造。结果表明,分子信标设计合理,性能稳定,其发夹结构在室温下既可成功闭合也可成功打开,最佳的活性鉴定条件为分子信标与适配体添加比例1.25∶1,孵育时间50 min,孵育温度为室温。活性位点分析表明Loop2-4是4种有机磷农药共有的活性位点,Loop2-3及SS4-54适配体5＇端和3＇端残余的核苷酸是甲拌磷重要的活性位点,Loop2-2和Loop4-2是丙溴磷和水胺硫磷共有的活性位点,Loop4-3是丙溴磷和氧化乐果共有的活性位点,Loop2-1和Loop4-1是水胺硫磷重要的活性位点。通过基因拼接改造的SS24-PJ-35适配体对丙溴磷和水胺硫磷的结合活性明显提高。

英文摘要：

A competitive inhibition method based on a molecular beacon containing 20 nucleotides was developed for the active site analysis and modification of two organophosphorusate pesticides aptamers that had been selected in our previous study.The results indicated that the designed molecular beacon formed a hairpin structure,and the loop could be opened up in the presence of complementary aptamer sequence.The ratio of molecular beacon to aptamer of 1.25 to 1,incubated time of 50 min and incubation at room temperature were chosen as optimal conditions of active site analysis.Under optimal conditions,the Loop2-4 was a mutual active site for four organophosphorus pesticides,Loop2-3 and the nucleotides at 3′ and 5′ of the SS4-54 aptamer were important active sites for phorate,Loop2-2 and Loop4-2 were mutual active sites for profenofos and isocarbophos,Loop4-3 was a mutual active site for profenofos and omethoate,Loop2-1 and Loop4-1 were important active sites for isocarbophos.The binding activity for profenofos and isocarbophos of SS24-PJ-35 aptamers modified by gene splicing significantly increased.