基于赭曲霉毒素A的高密度分子模拟表位的构建及其应用研究

项目名称： 基于赭曲霉毒素A的高密度分子模拟表位的构建及其应用研究

项目编号： No.30860240

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 刘仁荣

作者单位： 江西科技师范大学

项目金额： 26万元

中文摘要： 竞争免疫分析方法在赭曲霉毒素A、黄曲霉毒素B1等真菌毒素的快速检测中发挥着重要的作用，但该类方法须使用真菌毒素与载体偶联制备竞争抗原，给试剂的生产和使用者带来毒性危害。研制这些毒素的模拟表位、以之建立无毒害的免疫学检测方法具有较大的科学价值和应用前景。本研究通过噬菌体随机肽库技术，以赭曲霉毒素A单克隆抗体为靶标，成功筛选到11种模拟表位；但这些模拟表位都是表达在次要衣壳蛋白上，拷贝数量太少，无法将其直接应用于竞争抗原的制备。本研究通过分子生物学方法，应用PC89噬菌粒载体，将赭曲霉毒素A的模拟表位导入至M13噬菌体的主要衣壳蛋白pⅧ#19978;，并引入了肠激酶切割位点，通过单克隆抗体结合及DNA测序验证，获得了在N末端高密度表达模拟表位的重组噬菌体；再以其为竞争抗原，建立了新的竞争酶联免疫吸附检测（ELISA）方法，并对该方法的各项技术指标进行综合测试和对比实验；研究证明该方法在检测的灵敏度、准确度和精确度等方面与传统ELISA方法一致，并已经成功将该方法应用于建立黄曲霉毒素B1的竞争ELISA检测方法，本研究为类似的有毒物质的免疫学检测方法探索了一条高效、低成本、无毒害的制备竞争抗原的新方法。

中文关键词： 噬菌体肽库；酶联免疫分析；赭曲霉毒素A；模拟表位；噬菌粒

英文摘要： Immunoassay is an important method for detecting ochratoxin A (OTA) and aflatoxin B1 (AFB1).It involves in using the toxic mycotoxin in a conjugated form,which may be toxic to manufacturers and users. An promising approach to avoid using the toxic conjugated mycotoxin is to develop protein or peptide mimotope which mimic the mycotoxin.To replacing the toxic conjugate with mimotope in immunoassay, 11 mimotope peptides of OTA were acquired by a panning-elution selection from a phage random 7-peptide library. Since those mimotope were displayed on the N terminus of the minor coat protein gⅢ of the filamentous phage M13, the copy number of the expressed mimotope was too low to being used in the immunoassay. To solve such problem, an OTA mimotope peptide was fused with the major coat protein gVIIIp of the filamentous bacteriophage by a pC89 phagemid display system. It led to the expression of abundant recombinant gVIIIp chimeric proteins in mosaic bacteriophages. A cleavage site of enterokinase was introduced into the fusion gVIIIp, which allowed the OTA mimotope to be displayed on the N-terminus of the coat protein gVIIIp after cleavage. The recombinant phages bearing OTA mimotope peptides were identified by an anti-OTA monoclonal antibody, and confirmed by DNA sequencing. An indirect competitive ELISA was set up with the recombinant phage, and the new method was compared to a conventional ELISA. The calibration curves and the results of accuracy and precision were almost identical in both methods.The approach has been applied in the OTA ELISA successfully. The approach is promising for those haptens which are unstable, difficult to conjugate, valuable, dangerous, or toxic in practical applications.

英文关键词： phage library; ELISA; Ochratoxin A; Mimotope; phagemid