基于iTRAQ-LC-MS/MS的梨火疫病和亚洲梨火疫病特异性诊断靶标的发掘

Fire blight and fire blight of asian pear are important quarantine pest in China, the immunological detection and identification method based on antigen-antigen has a unique advantage in the field of on-site inspection and quarantine. Due to the high affinity of two pathogenic bacteria, it is difficult to establish specific immune detection method. The antiserum that previously obtained including the antibody which could specifically recognize the two pathogenic bacterias. However, it is too hard to purification of antiserum or screening of specific monoclonal antibody due to the specific diagnostic targets of two kinds of disease are not clear. This project designed to extract proteins from two diseased tissues for 2-DE separation, using Western Blot to screen candidate targets of the two diseases based on the available high titer antiserum against two kinds of pathogenic bacteria and carry out the iTRAQ-LC-MS/MS analysis. Simulation the epitopes of the target protein that have high expression and difference, also the molecular cloning and expression of the target protein’s gene were performed. Furthermore, the target induction effect of epitopes and expressed protein to specific antibodies would be studied. Finally, the specific diagnosis target for fire blight and fire blight of Asian Pear would be confirmed. The project will provide the foundation for rapid immunological diagnosis for fire blight and fire blight of Asian Pear; also provide the scientific guidance for efficient and accurate excavation of the specificity diagnosis target for plant disease.

梨火疫病和亚洲梨火疫病是我国重要检疫性有害生物，基于抗原抗体特异性结合的免疫学检测鉴定方法在其现场检验检疫中具有独特优势，但两种病原菌亲缘性高，难以建立特异性免疫检测方法。申请人前期 获得的抗血清中存在特异性识别两种病原菌的抗体，但两种病害特异性诊断靶标不明确，难以对抗血清进行纯化或筛选特异性单克隆抗体。本项目拟提取两种病组织全蛋白进行2-DE分离；通过Western Blot应用两种病原菌的抗血清对全蛋白进行识别，筛选出两种病害的候选靶标蛋白，进行iTRAQ质谱分析；对高峰度、差异性候选靶标蛋白进行抗原表位模拟，同时对候选靶标蛋白基因进行分子克隆、表达；研究模拟表位/表达蛋白对特异性识别两种病原菌的高亲和力抗体的靶向诱导效应，最终明确梨火疫病和亚洲梨火疫病的特异性诊断靶标。项目的顺利实施将会为梨火疫病和亚洲梨火疫病现场快速免疫诊断奠定基础；为植物病害特异性诊断靶标的发掘提供科学指导依据。

梨火疫病和亚洲梨火疫病是我国重要的检疫性有害生物，也是目前我国的一类农作物病害，因此在田间对梨火疫病菌和亚洲梨火疫病菌的快速、准确筛查是对两种病害执行检疫措施的重要前提。基于抗原抗体特异性结合的免疫学检测鉴定方法在现场检验检疫中具有独特优势。本研究通过分析梨火疫病菌和亚洲梨火疫病菌的全蛋白信息，发掘两种病原菌的特异性检测靶标（抗原），获得了可特异性识别梨火疫病菌和亚洲梨火疫病菌的高效单克隆抗体和多克隆抗体，从而确定了两种病原菌的特异性靶标，解析了特异性靶标位点和抗体筛选的关系，初步探索了特异性识别两种病原物的夹心式免疫检测体系，为梨火疫病菌和亚洲梨火疫病菌的现场快速筛查奠定了基础。