基于纳米抗体可变区CDR3的抗Cry2A多肽的识别机制研究

Bt Cry2A toxin is one of the most widely used genetically modified insecticides and biopesticides, as the use increases so does exposure to the Cry2A toxin. However, the study on immunoassay for Cry2A toxin is still relatively lagging behind. The preparation of new recognition elements is the basis and the primary limiting factor for the development of immunoassays. This project intends to focus on the preparation of recognition elements based on the nine anti-Cry2A nanobodies obtained previously. Using the co-exist amino acids in CDR3 of anti-Cry2A nanobodies as the core sequence, a phage display 12-peptide library is constructed for anti-Cry2A peptides screening. Thereafter, the positive anti-Cry2A peptides is identified, and the interaction mechanism of peptide and Cry2A with the application of computer-assisted molecular modeling is studied. By revealing the structural characteristics and intermolecular force characteristics of high affinity peptides, elucidating the recognition mechanism of anti-Cry2A peptide (key amino acids) and Cry2A, providing theoretical basis for rational design and directional modification of peptides and promoting the development of immunoassays for Cry2A toxin.

Bt Cry2A毒素是常见的转基因杀虫成分和生物杀虫剂之一，随着使用量的持续增加，Cry2A毒素的暴露问题日益严重。然而，目前针对Cry2A毒素免疫学检测方法的研究还相对滞后，新免疫识别元件的获得是其检测方法发展的前提和首要限制因素。本项目在前期获得9株抗Cry2A纳米抗体的基础上，围绕免疫识别元件的进一步制备，拟以抗Cry2A纳米抗体可变区CDR3的共同氨基酸作为核心序列，构建噬菌体展示十二肽偏向文库，定向筛选抗Cry2A识别多肽。对得到的识别多肽进行序列分析、特异性和亲和性分析，再辅助计算机分子模拟技术，进行识别多肽与Cry2A分子互作的分析，揭示高亲和性识别多肽的关键结构特征和分子间作用力特征，阐明识别多肽（关键氨基酸）与Cry2A的识别机制，为指导识别多肽的理性设计与定向改造提供理论依据，为促进Cry2A毒素免疫学检测方法的发展提供基础。

Bt Cry2A毒素是常见的转基因杀虫成分和生物杀虫剂之一，随着使用量的持续增加，Cry2A毒素的暴露问题日益严重，对农产品及环境中的Cry2A毒素进行有效监控十分必要。然而，目前针对Cry2A毒素免疫学检测方法的研究还相对滞后，新免疫识别元件的获得是其检测方法发展的前提和首要限制因素。本项目以Cry2A为靶分子，从天然纳米抗体库中筛选获得9株抗Cry2A纳米抗体，基于噬菌体展示纳米抗体P2，建立了双抗夹心化学发光分析法；随后对纳米抗体进行原核表达和纯化，基于纯化纳米抗体和量子点荧光微球探针，建立了双抗夹心荧光免疫分析法。所建立的方法具有高灵敏度、高特异性，能够用于谷物样品中Cry2A毒素的快速检测。纳米抗体CDR3区关键氨基酸在与目标物结合中起到关键作用，将纳米抗体CDR3区来源的多肽化学合成后进行活性鉴定和分子模拟，结果显示失去了FR区后，CDR3区氨基酸序列的空间结构发生改变，从而影响多肽的结合活性。本项目探究了纳米抗体在Bt Cry2A毒素免疫分析检测中的应用，初步探索了纳米抗体CDR3区氨基酸的构效关系，为指导纳米抗体和多肽的理性设计，促进新型免疫识别元件在分析诊断领域的应用提供基础。