基于高硼亲和力磁性纳米粒子的糖肽类抗生素适配体筛选方法研究

Glycopeptide antibiotics are a class of antibiotics containing cis –diol with similar structure and properties. This class of antibiotics residues in food can cause human body resistance to drugs, and make the human face "no medicine can cure" risk, so many countries have strictly controls of this class of antibiotics production, and it is forbidden to use in animal feed and aquatic feed. Aptamers, called as artificial antibodies, are an emerging class of molecules for molecular recognition with strong affinity and high specificity. Functionalized magnetic nanoparticles have advantage of easy modification, simple operation and high separation efficiency. Integration of aptamers and functionalized magnetic nanoparticles, a universal glycopeptide antibiotics aptamers selection method will be developed, which is helpful for specific, sensitive and rapid detection of antibiotics in food or feed. Based on the above assumptions, in this project, high-affinity boronate functionalized magnetic nanoparticles will be designed and prepared. Then integrated with this material and capillary electrophoresis, a universal glycopeptide antibiotics aptamers selection method will be established. We will focus on the effects of material surface modification, spacer and boronic acid species on affinity and specificity of the materials. The factors that influence the selection of aptamers, such as selection of DNA library, PCR amplification conditions, and the conditions for measurement of the affinity and specificity of aptamers with capillary electrophoresis - laser induced fluorescence will be analyzed. The successful implementation of this project has a positive significance for improving the glycopeptide antibiotics detection method, and ensuring the safety of animal food quality.

糖肽类抗生素是一类含有顺式二羟基、结构和性质相似的抗生素，其在食品中的残留会导致人体产生耐药性，使人类面临“无药可医”的危险境地。因此各国都严控该类抗生素的生产，严禁在动物饲料、水产饵料中使用。被称为人工抗体的核酸适配体是一类新型识别分子，亲和力强、特异性高；功能化磁性纳米粒子具有修饰方便、操作简单、分离效率高的优势；将二者结合，发展一种糖肽抗生素适配体筛选的普适方法，有助于食品或饲料中该类抗生素特异、灵敏、快速检测。本项目首先设计、制备高硼亲和力磁性纳米粒子，然后将该材料与毛细管电泳联用发展一种糖肽抗生素适配体筛选的普适方法；研究表面修饰，间隔臂及取代硼酸的种类对材料亲和力和特异性的影响；分析DNA文库选择，PCR扩增条件优化及毛细管电泳-激光诱导荧光测定适配体亲和力和特异性的条件和方法等影响适配体筛选的因素。本项目的实施对提升糖肽类抗生素检测水平，保障动物食品质量安全，具有积极意义。

糖肽类抗生素是通常用于治疗耐药感染的最后手段。其在食品中的残留会导致人体产生耐药性，使人类面临“无药可医”的危险境地。因此各国都严控该类抗生素的生产，严禁在动物饲料、水产饵料中使用。被称为人工抗体的核酸适配体是一类新型识别分子，亲和力强、特异性高，在分子识别、检测方面发挥了重要的作用，筛选糖肽类抗生素的适配体对于糖肽类抗生素的检测和识别具有重要的意义。高硼亲和力磁性纳米粒子由于对顺式二羟基化合物亲和力高，容量大，而备受关注。糖肽类抗生素是一类含有顺式二羟基、结构和性质相似的抗生素，因此采用高硼亲和力磁性纳米粒子预固定糖肽抗生素，发展一种糖肽抗生素适配体筛选的普适方法，对于食品或饲料中该类抗生素特异、灵敏、快速检测具有重要意义。本项目首先设计、制备了高硼亲和力磁性纳米粒子，然后将该材料与毛细管电泳联用发展了一种糖肽抗生素适配体筛选的普适方法；我们选择万古霉素为糖肽抗生素的模型分子，研究结果表明，经过一轮的高硼亲和磁性纳米粒子筛选，然后进行五轮毛细管电泳筛选，可以得到解离常数为微摩尔的万古霉素的适配体。该方法筛选速度快，得到的适配体亲和力和专一性好，而且避免了负筛选。该方法还可以用于其他顺式二羟基化合物，如血凝素HA和HA1的筛选。