脱氧雪腐镰刀菌烯醇抗原设计及直接竞争酶联免疫方法

发布时间：2018-02-01 18:31

  本文关键词： 脱氧雪腐镰刀菌烯醇(DON) 连接臂 分子模拟 直接竞争酶联免疫分析　出处：《江南大学》2011年硕士论文　论文类型：学位论文

【摘要】：脱氧雪腐镰刀菌烯醇(DON)半抗原的分子设计是建立其免疫分析方法的关键步骤,目前,DON半抗原设计仍采用经验法,造成时间和资金的浪费。本文从半抗原设计的最大原则(不改变待分析物的结构性质)出发,明确DON最佳半抗原的设计思想:最大化抗原表位相似度。利用分子模拟技术,通过分析分子的空间构型、电子特性、疏水性质这几个影响抗原-抗体间作用力的因素在DON修饰前后的变化,研究半抗原结构与免疫识别之间的关系,建立DON半抗原设计的理论模拟方法,为小分子半抗原设计提供新的思路,并在此基础上建立了DON的直接竞争酶联免疫分析方法。 根据DON抗原设计流程:选择结合位点→引入连接臂→偶联载体蛋白,首先研究结合位点对DON免疫识别的影响,在DON分子中的4个潜在结合位点(3 ,7 ,15位羟基以及8位酮基)引入相同碳链长度的的连接臂,借助分子模拟软件Hyperchem7.5对DON及半抗原的表位决定参数(空间结构、原子点电荷、疏水常数)进行计算分析,3位半抗原具有与DON最相似的结构性质,表明3位是DON引入连接臂的最佳位点,这与Casale和Ramesh的免疫实验结果一致。 其次,在3位羟基上设计不同的连接臂结构,通过抗原表位相似度模拟筛选得到最佳连接臂结构:丁二酸酐臂。同时合成不同连接臂的DON完全抗原,并将之进行免疫抗体实验,测定抗体的效价以及抑制率,验证分子模拟结果,研究半抗原结构对抗体免疫活性的关系。结果表明B组(顺丁烯二酸酐臂)抗体平均效价:256000,A组(丁二酸酐臂)、C组(邻苯二甲酸酐臂)平均效价:64000; DON浓度为500 ng/ml时对抗体的抑制率分别:A组(66.4 %)B组(21.1 %)C组(10.8 %)。抑制率的大小与抗体对DON的特异性相关,A组与DON具有最高的抗原表位相似度,抗体对DON的特异性强,抑制率高;抗体的效价可以反映半抗原分子的抗原性,苯环结构、不饱和键可以增强抗原性,B组连接臂中含有不饱和键,增强了半抗原的抗原性,产生的抗体效价高;C组虽含有苯环结构,确未能产生高效价的抗体,结合其空间结构发现C组半抗原中羧基端与半抗原主体结构非常靠近,半抗原结构可能被载体蛋白屏蔽而影响免疫识别;综合考虑,丁二酸酐臂是DON的最佳连接臂,免疫试验结果与分子模拟具有很好的相关性。 在最佳半抗原基础上,比较BSA和KLH两种载体蛋白的DON完全抗原的免疫原性。通过丁二酸酐衍生法和碳二亚胺法(EDC法)制备不同载体蛋白的DON完全抗原,免疫新西兰长耳兔获得DON抗体,并运用酶联免疫方法(ELISA)对制备的抗体进行研究。 在上述研究结果基础上合成DON-KLH完全抗原,建立DON的直接竞争酶联免疫分析方法,通过一步抗原抗体反应,快速检测食品中DON。检测范围为1 -100 ng/mL。半数抑制率IC50为10 ng/mL,最低检出限为0.56 ng/mL,与T-2毒素等真菌毒素的交叉反应率小于12 %;平均批内变异系数为2.82 %,平均批间变异系数为14.54 %;对玉米粉的加标回收率在80.2-91.1 %,准确性较高;与商品化试剂盒相比具有所需时间短,精密度高,灵敏度高的优点。 本研究表明分子模拟方法对DON半抗原设计有积极作用,指导半抗原设计,减少半抗原设计中时间及资金的投入。不同载体DON免疫抗原的免疫原性不同,DON-KLH免疫原性优于DON-BSA。6本研究所建立的DON的直接竞争ELISA方法,快速、方便,具有较好的实用价值,为进一步研究DON的ELISA检测试剂盒提供了重要的实验依据。
[Abstract]:Deoxynivalenol (DON) molecular design of the hapten is a key step to establish its immune analysis method, at present, DON hapten design still uses empirical method, resulting in the waste of time and money. This article from the principle of maximum hapten design (without changing the structure properties of the analyte) of clear design ideas DON best hapten: maximizing the epitope similarity. By using molecular simulation methods, the spatial configuration, analysis of molecular electronic properties, hydrophobic nature of these effects of antigen antibody interaction factors in the changes of DON before and after modification, the study of the relationship between hapten structure and immune recognition, a simulation method of DON theory antigen design, provide a new way of thinking for hapten design, and established a direct competitive enzyme linked immunosorbent assay DON.
According to the design process: introduction of DON antigen, connecting arm coupled to carrier protein binding sites, first, study the combined impact of DON sites of immune recognition, 4 potential binding sites in DON molecules (3, 7, 15 and 8 hydroxy ketone) connecting arm uses the same length of carbon chain, with the help of molecular simulation software Hyperchem7.5 of DON and semi epitope decision parameters (spatial structure, atomic charge, hydrophobic constant) were calculated and analyzed, 3 semi antigen has the most similar structural properties with DON, showed that the 3 is DON into the best sites of the connecting arm, and the experimental results of immune Casale and Ramesh.
Secondly, the design of connecting arm structure in different 3 hydroxy group, through epitope screening the optimal similarity simulation connecting arm structure: Ding two anhydride arm connecting arm. At the same time, the synthesis of different DON antigen, and the immune antibody test, determination of antibody titer and inhibition rate, simulation results verify the molecular research. Relationship of hapten structure and immune activity. The results showed that group B (maleic anhydride arm): 256000, the average antibody titer of A group (d two C group (anhydride arm), phthalic anhydride two formic acid arm) average value: 64000; 500 DON concentration of ng/ml to inhibit the antibody respectively. Group A (66.4%) B (21.1%) C group (10.8%). The size and inhibition rate of antibodies specific for DON, A and DON have the highest similarity of antibody epitopes, the specificity of strong DON inhibitory rate; antibody titer can reflect the antigenicity of hapten molecules, The benzene ring structure, unsaturated bond can enhance the antigenicity of B group, the connecting arm containing unsaturated bonds, enhance the antigenicity of hapten, the high antibody titer; although group C containing benzene ring structure, it failed to produce high titer antibody, combined with the spatial structure of C group in the end carboxyl hapten with the hapten subject very close to the structure, structure of hapten carrier protein may be shielded by immune recognition; comprehensive consideration, Ding two anhydride arm is the best connecting arm of DON, simulation and molecular immunity test results have a good correlation.
In the best hapten based on the immunogenicity of two carrier proteins BSA and KLH DON complete antigen. By two D anhydride derivatization and carbon two imide method (EDC method) were prepared with different carrier protein DON antigen, immune New Zealand rabbits received DON antibody, and using ELISA method (ELISA) to study the preparation of antibody.
鍦ㄤ笂杩扮爺绌剁粨鏋滃熀纭，

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