【作者】 王明华；

【导师】 王剑平；

【作者基本信息】 浙江大学 ， 生物系统工程， 2011， 博士

【摘要】 动物性食品的营养丰富且易于人体消化吸收,具有极高的营养价值,随着社会和科技的发展,动物性食品的消费需求迅速增长。动物性食品生产依赖于畜牧养殖业的发展水平,先进的遗传育种与养殖技术为提高动物性食品产量提供了前提条件的同时又带来了人们更加关注的食品安全问题,兽药残留问题尤为突出。免疫传感器作为一种检测分析方法,主要以其快速简便的优点,得到了研究和应用领域的广泛关注,其从研究到应用的关键是要获得传感器稳定的性能表现。生物传感器所倚赖的特异性生物反应本身以及生物分子与传感器结合,都极易受到环境因素的影响,从而影响其性能。分子模拟是依赖于量子力学、分子力学用计算机来构造、实现、分析和存储复杂的分子模型,计算微观粒子之间的相互作用,在分子或原子水平上进行的科研。该方法具有成本低、安全性高、可深入研究问题等优点,在众多研究领域体现出了应用潜力。本研究以动物性食品安全问题中的青霉素兽药残留快速检测免疫传感器为研究目标,主要关注直接固定抗体直接检测方法的性能稳定性。针对该方法中固定抗体的活性和稳定性确证,采用分子模拟的研究方法对其可能的影响操作步骤和检测条件——牛血清蛋白封闭的过程进行深入探索,以期改进免疫传感器的构建方式,有助于获得较稳定的性能。免疫传感器采用硫辛酸自组装单分子层膜将氨芐青霉素抗体固定于压电金电极上作为生物识别元件、以石英晶体微天平作为检测器构建,对不同封闭过程产生的影响进行研究。比较免疫传感器实物试验与分子模拟的研究结果,同时,也对分子模拟在生物传感器研究和应用中其他建立和检测步骤的应用可行性进行评价。主要研究内容如下：(1)选择了三种基于网络平台的不同蛋白-蛋白相互作用模拟预测方法FiberDock (http://bioinfo3d.cs.tau.ac.il/FiberDock), ClusPro(http://cluspro.bu.edu/), GRAMM-X (http://vakser.bioinformatics.ku.edu/resources/gramm/grammx),对氨芐青霉素抗体活性部位与人血清蛋白、卵白蛋白之间的相互作用进行模拟。这三种方式都可以对含有多条肽链的蛋白质之间的相互作用完整表现。结果表明,对于本研究中氨芐青霉素抗体与HSA、OVA相互作用的模拟预测,三种分析方法都可用于模拟研究,结果均表明了氨芐青霉素抗体与HSA、OVA有发生非特异性相互作用的可能性。三种方法的模拟研究结果之间具有一定相似性和一致性。(2) ZDOCK程序是一种采用快速傅里叶变换算法的蛋白-蛋白相互作用模拟研究程序,可对不同蛋白单条多肽链之间的相互作用进行研究。对包含抗体活性中心的A链与人血清蛋白A链、卵白蛋白A链,以及不含抗体活性中心的抗体B链与人血清蛋白A链之间的相互作用进行模拟,并与溶菌酶及其抗体之间的特异性相互作用模拟结果进行比较分析。模拟研究结果表明抗体活性中心所在的A链与人血清蛋白A链发生非特异性相互作用的几率大于B链和卵白蛋白A链,这可能与抗体制备过程采用牛血清蛋白作为氨芐青霉素偶联蛋白有关。与溶菌酶及其抗体的特异性相互作用模拟结果对照表明,蛋白-蛋白相互作用模拟预测方法可用于深入了解抗体的特异性。(3)将氨芐青霉素抗体序列与蛋白质数据库PDB中收入的其它抗体序列通过BLAST序列比对氨芐青霉素抗体的抗原结合区域进行了确证,结果表明氨芐青霉素抗体A链模拟结果构象中参与形成对接复合物的区域,大部分是位于抗体结构中的高度可变区域中与抗体特异性密切相关的关键氨基残基。(4)对硫辛酸自组装单分子层膜将抗体固定于压电金电极表面作为生物识别元件、石英晶体微天平为检测器的氨芐青霉素检测免疫传感器建立过程中,固定抗体后采用不同浓度、时间的牛血清蛋白封闭进行研究。结果表明固定抗体后采用牛血清蛋白进行封闭,修饰电极表面结合牛血清蛋白的质量表现出了浓度和时间的依赖性；电极表面的活化羧基连接位点上,牛血清蛋白结合量不随固定抗体质量增加而减少,表明氨芐青霉素抗体与牛血清蛋白之间有发生非特异性相互作用的可能性。(5)对采用不同试剂和牛血清蛋白不同时间、浓度封闭处理的免疫传感器进行了检测氨芐青霉素试验。结果表明免疫传感器构建中,采用BSA封闭处理时,BSA与抗体修饰电极结合量较少的处理,其对氨芐青霉素的检测性能优于BSA结合量较多的处理。更多还原

【Abstract】 With the development of modern animal husbandry and nutritional knowledge, the requirement of animal derived food is becoming more and more highlighting. The nutrients of animal derived food are abundant, they tend to be assimilated and digestedeasily by human. The produce of animal derived food is based on the technology of modern animal husbandry,usage of molecular biosciencesbreeding andcultivating techniquemore and more guarantees livestock-bird growth healthy and the animal husbandry development regularly. On the other hand, unreasonable added beast medicine easily brings aboutveterinary medicine residueproblem of animal derived food safety.As an analytical approach,immunosensor is widely used and studied for its simple-design, high-sensitivity and low-cost. Although there are many research findings, practical application in the market is few mainly for the unreliable performance.The biological recognition and biomolecules immobilization were lots of environmental sensitivity,and effect on the immunosensor performance.Molecular simulation is a molecular lever research approach based on quantum mechanics (QM), molecular mechanics (MM) and strong computation capability of advanced computer. Because of the advantage of decreasing research cost, strengthened safety, detailed knowledge and so on,computational studies have beenused in many different fields.This work aimed to the developmentof penicillin residue detection immunosensor for animal derived food safety, and mainly concerned to the mode of direct detection by immobilized antibody. In order to well understand the efficiency of immobilized antibody,possible reasons of biosensor building and detecting, the BSA blocking, was studied by molecular simulation running on computer. The immobilization of antibody is based on self-assembly monolayer of thioctic acid formed on electrode surface.Different BSA blocking modes were investigatedby detecting the signal withpiezoelectric crystal microbalance.Evaluate and discuss the molecular simulation application possibility of immunosensor research.The main contents and results are summarized as follows.(1) Three different web served protein-protein interaction simulation methods had been used in the study for ampicillin antibody specific evaluation. ClusPro(http://cluspro.bu.edu/), GRAMM-X (http://vakser.bioinformatics.ku.edu/resources/gramm/grammx), FiberDock (http://bioinfo3d.cs.tau.ac.il/FiberDock). These programsprovided an overall interaction of ampicillin antibody and HSA/OVA with similarity and consistency, indicated that ampicillin antibody complementarity determining region is possibly to bind to HSA/OVA in biosensor building process and detection samples.(2) ZDOCK simulation used to further evaluation of docked protein-protein complexes. By contrast with the specific interaction between antibody and antigen (lysozyme and antibody), it is found that the cluster density of non-specific interaction docking complexes is smaller, dock score of these poses are more scattered too. However, non-specific interaction of ampicillin antibody chain A and HSA presented a higher score than lysozyme and antibody, but chain B and HSA given a reasonable low score. Duing to the solvation effects electrostatic and Van der Waals interactions are important to form biological molecules and complexes, including in immune reaction. The formation probability of antibogy-OVA docking complex was lower than antibogy-HSA implying that antibody preparing process may impact on its specific.(3) To analysis the binding complexproperties,the amino acid sequence of ampicillin antibody and other PDB 3D antibody structurewere aligned by BLAST program. The result shown that almost all curial crucialamino acid residues of ampicillin chain A were located in the complex surface.(4) The result of experimental study on different BSA blocking shown that the mass of adsorbing BSA on antibody modified electrode performing concentration and time dependence, mass of immobilized antibody don’t displaynegative correlation with BSA mass. It is indicated that the interaction possibility of ampicillin antibody and BSA.(5) Electrochemical characteristics of the immobilization and immune response of different immunosensors with various immobilized antibody and blocking reagentwere determined by cyclic voltammetry. The result showed that the building schema of immunosensor is successful. In addition, cyclic voltammetry (CV) was employed to realize the different blocking processed electrodesdetection of pesticide residues.The more bounded blocking BSA associated, the less ampicillin detected.更多还原