禽波氏杆菌血凝素的宿主互作蛋白筛选及其功能研究

Bordetella avium (B. avium), one of the most important bacterial pathogens, causes a highly contagious infectious upper respiratory disease of poultry. It was also isolated from patients with lung disease in recent years. Previous studies have found that hemagglutinin correlates completely with the ability of a B. avium strain to colonize in trachea and cause disease in host. It’s urgent to reveal its interacting host protein and interaction with host cells. In this study, The cDNA bank of host trachea cells infected by B. avium standard strain with full hemagglutination activity would be built, and the special interacting protein and coding gene would be screened by the method of yeast two hybrid to obtain target genes of hemagglutinin. Interacting protein structure prediction, interaction domains screening and identification of organ-specific expression would be done. Then the hemagglutinin colonization environment within host cells would be revealed by monitoring the role region of hemagglutinin and interacting protein in cells. To study the interaction between hemagglutinin and its interacting protein, hemagglutinin expression differences between in vivo and in vitro would be monitored, as well as interacting protein expression differences between in standard strain infected chicks, mutant strain infected chicks and healthy chicks. And hemagglutinin-interacting protein complexes would be detected by co immunoprecipitation assay. At last, antibody blocking assay would be done to evaluate if B. avium colonization in trachea could be blocked by antibody. The purpose of the study is to discover the pathogenic basis of B. avium. It can also provide scientific basis for regulation of target genes, improving immune protection of host and blocking colonization and infection of B. avium.

禽波氏杆菌是导致禽类高度接触传染性上呼吸道疾病的重要细菌病原，近年来在人类肺病患者体内也分离到了该菌，而血凝素与其黏附气管细胞并导致疾病的能力直接相关。本项目拟构建完全凝集活性禽波氏杆菌标准株感染鸡气管的cDNA文库，酵母双杂交筛选血凝素与气管细胞发生特异性作用的相关蛋白及其编码基因，获取血凝素在宿主体内的靶基因；预测互作蛋白结构并筛选互作结构域，监测血凝素与互作蛋白在细胞中的作用区域，并鉴定互作蛋白表达的器官特异性，揭示血凝素作用的宿主细胞内环境；监测血凝素在宿主体内外的表达特征差异和互作蛋白在标准株感染鸡、突变株感染鸡和健康鸡体内的表达差异，免疫共沉淀检测血凝素-互作蛋白的作用复合物，对禽波氏杆菌在气管细胞中的定植进行抗体阻断，评价阻断效果，探讨二者在宿主体内的相互作用。旨在揭示禽波氏杆菌的致病基础，为进一步研究靶基因的调控，提高宿主免疫保护力并阻断禽波氏杆菌的定植和感染提供科学依据。

禽波氏杆菌是导致禽类高度接触传染性上呼吸道疾病的重要细菌病原。该项目对近年来禽波氏杆菌病开展了流行病学调查，继而从实验室多年来保存的和新分离的禽波氏杆菌菌株中筛选出强凝集活性菌株用于项目研究；通过对凝集相关基因hagA 和hagB 分别进行克隆和抗原表位预测分析，表明Hag A蛋白氨基酸存在7个优势B细胞抗原表位、6个可能的糖基化位点，Hag B蛋白氨基酸存在18个优势B细胞抗原表位、15个可能的糖基化位点，且可能存在未知的特殊高级结构；确定了培养基、温度、pH值等不同培养条件对hagA 和hagB表达的影响；经基因表达、抗体制备、动物实验、抗体阻断等实验发现禽波氏杆菌在宿主气管中的定殖可以被hagB-2蛋白抗体部分阻断，表明二者在宿主体内存在相互作用；构建了强凝集活性禽波氏杆菌菌株感染雏鸡气管的cDNA文库；以hagB-2表达产物作为诱饵蛋白，经酵母双杂交筛选到了血凝素与气管细胞发生特异性作用的相关蛋白；经免疫酶组化和病理学监测确定互作蛋白的表达具有器官特异性，二者在宿主体内的相互作用区域位于气管细胞表面和胞浆中；同时检测了血凝素与互作蛋白结合后禽波氏杆菌对雏鸡气管和肺脏的定殖和病变规律，推测互作蛋白与血凝素结合后介导了禽波氏杆菌在细胞表面的定殖和进一步的感染。