鸭源鸡杆菌菌毛编码基因的鉴定及其结构基因功能解析

Gallibacterium anatis(G.anatis) is the type species of Gallibacterium which is grouped as a new genus within the family Pasteurellaceae. The recent research results show that it's the main cause of salpingitis and oviduct cysts for infected layers.Up to now,there is a few report about its pathogenic mechanism, genomic constitution etc. The pathopoiesis of bacteria include multiple mechanisms and the complex process, among them the bacterial adhesion to host cells is the first key step to pathogen invasion, and fimbria is the adherent structures first discovered. The presence of fimbriae on the surface of G. anatis were discovered recently,but the molecular characteristics and exact role of them remains unclear.The proposed study will identify the genes encoding fimbriae of G.anatis by bioinformatics based on the finished genome sequence of G.anatis strain YU-ZZ-GX-3-DG isolated from China,and certify the functions of its structure genes by Gene knockout，animal infections and cell adhesion in vitro. The study will be the base to reveal the molecular characteristics, biological function, cell receptor binding domain of fimbriae of G.anatis and study the molecular pathogenesis mechanism of interaction between the fimbriae and the susceptible host cells, and so on.

鸭源鸡杆菌是巴氏杆菌科鸡杆菌属的代表种，为鸡输卵管囊肿和腹膜炎的重要病因。迄今为止，关于该菌的致病机制、基因组组成等的相关研究甚少。细菌的致病包括多重机制和复杂过程,其中细菌黏附于宿主细胞是病原菌入侵宿主关键的第一步，而菌毛是最早被发现具有重要黏附特性的菌体结构。新近发现鸭源鸡杆菌具有菌毛，但菌毛的分子特征及确切作用尚不清楚。本课题拟用生物信息学方法对本实验室已完成基因组测序的鸭源鸡杆菌中国分离株YU-ZZ-GX-3-QG进行序列分析，鉴定其菌毛编码基因；利用基因敲除技术、动物感染试验及体外细胞试验等对其结构基因进行验证及功能解析，为进一步揭示鸭源鸡杆菌的菌毛分子特征、生物学功能、细胞受体结合域的确定、与机体相互作用的分子致病机理以及构建基因工程候选菌苗等奠定基础。

鸭源鸡杆菌是鸡输卵管囊肿和腹膜炎的重要病因。迄今为止，关于该菌的致病机制、基因组组成等的相关研究甚少。细菌黏附于宿主细胞是病原菌入侵宿主关键的第一步，而菌毛是最早被发现具有重要黏附特性的菌体结构。本课题首先通过生物信息学方法对本实验室已完成基因组测序的鸭源鸡杆菌中国分离株YU-ZZ-GX-3-QG进行序列分析，鉴定出573 bp的F17样菌毛编码基因flfA；并制备出用于后期菌株鉴定FlFA多抗,同时利用自然转化法与结合转移法最终成功构建F17样菌毛编码基因敲除菌株，对敲除菌株系列功能验证显示菌毛基因的缺失不影响菌落形态大小、生长速率在对数期有一定差异；缺失株不能形成完整的菌毛、生物被膜形成能力下降、对鸡原代输卵管上皮细胞黏附力下降、且相对于野生株，缺失突变株对原代鸡输卵管上皮细胞的损伤明显大大降低、对小鼠致病力明显降低等，本研究为进一步揭示鸭源鸡杆菌的菌毛分子特征、生物学功能、细胞受体结合域的确定、与机体相互作用的分子致病机理以及构建基因工程候选菌苗等奠定基础。项目资助发表中文一级学报8篇，申请专利1项，待发表英文文章2-3篇，中文文章3篇。培养博士、硕士生5名，其中3名已经取得硕士学位，2名在读。项目投入经费22万元，支出21.9887万元，各项支出基本与预算相符。剩余经费0.0113万元。