核糖体展示TALEs突变文库筛选食源性致病菌特征双链DNA特异模拟抗体的研究

PCR detection method is time-consuming with high false-positive rate and the immunoassay is low sensitivity for foodborne bacteria. Aimed to this situation, this project will carry out the following work. Hypervariable region of transcriptional activator-like effector (TALE) is randomized by site-directed mutagenesis technology and then ribosome display mutant TALEs library is constructed by introducing ribosome display components. Targeted specific double- stranded DNA of Escherichia coli O157, Vibrio parahaemolyticus and Shigella, the library was rapidly screened and high specific mimic antibodies against different bacteria would be obtained by multicycle pannings and would be functionally expressed in E. coli. Furthermore, the property of these obtained mimic antibodies (such as affinity, titer and binding sites or groups etc) would be evaluated and characterized by the surface plasmon resonance(SPR) and other technologies. Based on elucidating the molecular mechanism of the specific recognition, highly sensitive detection methods for foodborne pathogens is established by the specific recognition between the mimic antibody and double-stranded DNA. This project is an exploration of innovative application for TALEs to the field of preventive medicine. This work not only will present new detection materials for the monitoring of pathogens, but also will provide new genetic manipulation tools for molecular biology.

针对食源性致病菌核酸PCR法检测耗时长、假阳性率高，全菌免疫检测灵敏度低的问题，本项目拟利用基因定点突变技术对转录激活样效应因子（TALE）的高可变区进行随机化，重叠引物延伸法引入系列展示元件，构建核糖体展示TALE突变文库，以典型食源性致病大肠杆菌O157、志贺氏菌和副溶血弧菌等的特征双链DNA为靶标，通过多轮生物淘筛最终获得特异识别双链DNA的模拟抗体；进行功能表达与纯化后，系统分析模拟抗体的结合特异性、亲和力与结合位点等，初步阐明特异识别的分子机制，从而在蛋白-双链DNA的特异识别的基础上建立食源性致病菌的高灵敏检测方法。本项目是新型分子生物学工具在预防医学领域的创新性发展，属于分子生物学、免疫学和微生物学等多学科交叉的探索研究，不仅可为致病菌的监控提供新的检测材料和技术方法，而且还可为分子生物学领域提供全新的基因操作工具。

对转录激活样效应因子（TALE）的高可变区进行设计，利用DNA全合成技术构建了TALEs库，以典型食源致病菌大肠杆菌O157等特征双链DNA为靶标，通过多次设计筛选最终获得特异识别双链DNA的TALE抗体物；进行功能表达与纯化后，利用SPR等技术系统分析TALE的结合特异性、亲和力与结合位点等，在蛋白-双链DNA的特异识别的基础上建立食源致病菌的高灵敏检测方法如夹心ELISA和基于适配体的滚环扩增(RPA)技术，其中RPA的检测技术达到了5个数量级。本项目是新型分子生物学工具在预防医学领域的创新性发展，不仅可为致病菌的监控提供新的检测材料和技术方法，而且还可为分子生物学领域提供全新的基因操作工具。