抗独特型抗体技术制备具有Bt毒蛋白活性杀虫生物肽研究

Anti-idiotype antibodies are antibodies against the antigen binding site of another antibody.The antigen binding site of the anti-idiotype antibody is similar in structure to the original antigen. Therefore, anti-idiotypic monoclonal antibodies can be used to mimic the structure of the original antigen. This study was based on the principle that the anti-idiotype antibody can mimic specific molecular conformation of the antigen epitopes and also its biological activity. The idiotype antibodies of the Bt toxin proteins (Cry1Ab, Cry1B Cry1C) are used as templates to prepare anti-idiotype antibodies, which have similar biological binding activties with the Bt toxin proteins, by employing human antibody library select system. The bioinformatics methods are also used to analyse homologous genes sequence, and also the 3D structure of the active binding sites which interact between anti-idiotype antibody and specific insect targets receptor (BBMV). This is an early exploration to develop new safe protein pesticide. Besides the prokaryotic expression system, the insect cell expression system was also used to ensure biological effects of the anti-idiotype antibody. The BBMV binding activities and insecticidal activities of expression products are measured to compare with original Bt toxin protein. The active bio-peptides, which can mimic Bt toxin proteins biological activities, are used to alternative Bt toxin proteins for pest control. And their genes are used to make gene transformation and expression in rice. And also their transgenic application potential will be verified.

抗独特型抗体是指针对抗体分子的特异抗原表位群（独特型）的抗抗体，与原抗原的决定簇分子互为"内影像"关系,可模拟抗原结构和功能。本研究拟基于抗独特型抗体模拟特定抗原决定簇分子构像及其生物活性的原理，计划采用Bt毒蛋白（Cry1Ab、Cry1B及Cry1C）独特型抗体为模板，采用人源抗体库的抗独特型抗体筛选路线，制备获得具有模拟相应Bt毒蛋白结合活性的抗独特型抗体。并利用生物信息学方法分析比较抗独特型抗体蛋白基因同源序列及其与特异靶标受体(BBMV)相互作用的结合位点相关结构特点，为开发新型安全蛋白农药做前期探索。为保障其生物效应，拟在原核表达体系基础上，构建昆虫细胞表达体系。表达产物将进一步采用BBMV结合试验及生物学活性实验，与原Bt毒蛋白做生物效应比对。对与原Bt毒蛋白具有相当生物活性的生物效应物，可替代Bt毒蛋白用于害虫防治，并开展在水稻中基因转化和表达，验证其应用潜力。

利用Cry毒素单抗或多抗作为筛选分子，结合噬菌体抗体库技术，建立筛选制备Cry1Ab和Cry1C毒蛋白抗独特型抗体的高效生物筛选及鉴定技术体系，建立相应的昆虫真核细胞表达体系及农杆菌介导的转基因水稻技术平台。利用噬菌体展示技术，采用负筛选策略成功获得针对Cry1Ab和Cry1C的抗独特型单链抗体；并通过免疫学和生物学测定，确定其亚型为“Ab2β”。 将筛选并鉴定为“Ab2β”类型的抗独特型单链抗体片段，经NCBI数据库、通过swissmodel同源建模进行基因比对及蛋白3D结构分析，并通过基因工程手段，克隆到原核表达载体和植物双元表达载体，分别在大肠杆菌和水稻中进行蛋白表达，并对表达蛋白进行小菜蛾BBMV结合活性及杀虫生物活性测定，以验证该模拟Bt毒素的新型蛋白质的应用潜力。研究发现仅在噬菌体展示型（融合）表达形式下具有模拟Bt毒蛋白的结合活性及生物活性（72h致死率均超过50%），可溶性（原核及真核（昆虫及水稻））表达时丧失该结合活性。通过对不同抗独特型抗体及相应毒素的生物信息学分析比较，发现抗独特型抗体的轻链CDR2区、CDR3区和重链CDR3区与相应Bt毒蛋白的C端、middle及N端保守结构域具有相似结构，预测为抗独特型抗体的主要活性作用位点。针对杀虫生物活性较好的抗独特型抗体（展示型表达形式），对其进行基因工程改造，以检测抗独特型抗体重链区、轻链区的功能，获得具高结合活性的可溶性表达的改造抗独特型抗体。抗独特型抗体技术应用于替代Bt毒蛋白的活性杀虫蛋白，这在国内外均为前沿探索性工作，为首次开展，项目获得了我国首批新型安全人源抗虫基因（蛋白）资源。

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