目录
摘要Ⅰ
关键词Ⅰ
AbstractⅡ
引言
引言1
1材料与方法2
1.1实验材料与仪器设备 2
1.1.1实验材料2
1.1.2实验仪器2
1.2实验方法 2
1.2.1质粒构建2
1.2.2大肠杆菌BL21(DE3)电转感受态的制备以及质粒转化4
1.2.3蛋白表达、提取、纯化及验证5
1.2.4抗体对四种蛋白亲和力测定6
1.2.5基于BRET原理的HFIA的原理以及操作步骤6
1.2.6条件优化7
1.2.7标准曲线的建立7
1.2.8特异性7
1.2.9基质效应8
1.2.10添加回收8
1.2.11仪器相关性8
2结果与分析8
2.1BRET供体、受体以及模拟表位序列设计及分析8
2.2融合蛋白表达的鉴定9
2.2.1SDSPAGE鉴定9
2.2.2Westernblot鉴定10
2.3融合蛋白亲和力的测定10
2.4条件优化11
2.4.1荧光传感器与抗体浓度的选择11
2.4.2蛋白浓度优化11
2.4.3孵育温度与时间优化11
2.4.4甲醇含量12
2.5敏感性13
2.6特异性13
2.7基质效应14
2.8添加回收15
2.9仪器相关性16
3讨论 17
致谢17
参考文献19基于重组模拟表位的吡虫啉生物发光共振能量转移免疫分析
摘 要
吡虫啉是一种广泛使用的广谱、高效烟碱类杀虫剂。由于其大量使用，在环境和农产品中的残留会危害生态系统和人体健康。因此建立快速有效的吡虫啉残留检测方法有很大的意义。免疫分析方法由于其较高的灵敏度和较为便捷的检测流程，近年来应用越发广泛。农药等小分子的免疫检测一般采用竞争模式。利用噬菌体展示多肽技术筛选高质量异源竞争 *51今日免费论文网|www.51jrft.com +Q: @351916072@ 
物，污染少，并且获得的噬菌体易于大量制备和纯化，加快了异源免疫分析方法的建立。本研究利用噬菌体展示多肽技术获取的模拟表位，将其与纳米荧光素酶（NanoLuc）和黄绿色荧光蛋白（mNeonGreen）融合表达制备了重组模拟表位。利用生物发光共振能量转移（BRET）技术，以NanoLuc作为荧光供体，mNeonGreen作为受体制备免标记的荧光生物传感器。通过构建质粒，蛋白表达、提取、纯化及验证获得四种模拟表位排列顺序不同的荧光生物传感器Sensor1、Sensor2、Sensor3和Sensor4，通过比较这四种荧光生物传感器与抗体的亲和性，选择亲和性最高的Sensor1作为后续研究材料，建立了检测吡虫啉免标记的均相、一步法的生物发光共振能量转移免疫分析方法。通过对抗体、蛋白用量、孵育时间、温度及工作缓冲液进行优化，在最优条件下建立了检测吡虫啉的标准曲线，为吡虫啉的残留快速检测提供了一种新的可行性方法。
IMMUNOASSAY OF IMIDACLOPRID BIOLUMINESCENCE RESONANCE ENERGY TRANSFER BASED ON RECOMBINANT MIMOTOPE
ABSTRAT
Imidacloprid, a broadspectrum neonicotinoid insecticide, is widely used worldwide. However, owing to its significant use, the residue in environment and agricultural products can harm ecosystems and human health. Therefore, it is of great significance to develop rapid and efficient methods for the detection of imidacloprid residues. In recent years, immunoassays have become more widely uesd in pesticide residue detection with the advantages of higher sensitivity and more convenient detection process. The immunoassays with a competitive format are generally used to detect small molecular weight compounds (including pesticide). Phage display peptide technology can easily screen the best competitors and has the advantage of less pollution, and the isolated phage is easy to prepare and purify in large quantities, which accelerate the development of heterogeneous immunoassays. In this study, we fused the peptidomimetic that was isolated by phage display technology to NanoLuc and mNeonGreen to generate novel fluorescent biosensors, and designed a bioluminescence resonance energy transfer (BRET) systems by using NanoLuc as the fluorescent donor and mNeonGreen as the receptor. By the process of construction of the vector, expression, extraction, purification and verification of the recombinant fluorescent biosensors, we successfully prepared four fluorescent biosensors (Sensor1, Sensor2, Sensor3 and Sensor4) that the order of the phageborne peptide varied. After analyzing the relative affinity to antibody of the individual fluorescent biosensors, we selected Sensor1 that showed the highest affinity to develop labelfree, homogeneous, onestep immunoassay for the determination of imidacloprid by BRET. After optimizing antibody, protein dosage, incubation time, temperature and working buffer, the standard curve of imidacloprid detection was established under the optimal conditions, which provided a new method for the rapid detection of imidacloprid.

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