布鲁氏菌优势抗原表位的筛选及TIR结构域蛋白阻断宿主细胞Toll样受体信号途径的分子机制研究

Brucellosis caused by Brucella can spread between people and animals, and it was classified as the second class infectious disease in China. In last several years, the infection rate of Brucella appeared a rising trend in farming and animal husbandry region of Inner Mongolia autonomous region of our country. At the same time, the development of normal animal vaccines has been limited, due to the problem of strong toxicity returning in using process. To date, there is still no Brucella vaccine for human using. Therefore, the claim for developing effective Brucella vaccine for animals and people using is very urgent. In this project, the polymerization function of Brucella BLS molecule was taken as the foothold. By a series of research contents, including protein antigens fusion expression with BLS molecule, affinity purification and antibody preparation, Brucella advanced antigen epitopes were screened. And corresponding nucleic acid and protein subunit vaccines were prepared. At the same time, we will study the probable signaling pathway in animal macrophages, which is associated with Brucella advanced antigen epitope molecules, using pull-down and mass spectrometry technology. In addition, Brucella TIR domain-containing protein gene will be cloned, and its role in blocking Toll-like receptors signaling pathways will be studied in Drosophila S2 cell and human T293 cell using dual luciferase experiment. The binding function between Brucella TIR domain-containing protein and MyD88 molecule in Drosophila S2 cell or human T293 cell was also studied by pull-down and Co-IP technology. At last, we will provide some target points for a new vaccine manufacture.

布鲁氏菌病是由布鲁氏菌造成的可在人、畜之间传播的二类传染病，近几年在内蒙古农牧业地区的感染率出现了上升趋势；同时，因为使用过程存在毒性反强的问题，常规动物疫苗的发展受到了限制，至今没有可供人使用的布病疫苗，因此，研制行之有效的动物及人用布病疫苗的要求十分迫切。本项目以布鲁氏菌BLS分子自身聚合作用作为立足点，通过蛋白质抗原与BLS分子的融合表达、亲和纯化、抗体制备等一系列研究内容，筛选布菌优势抗原表位分子并制备相应的核酸和蛋白质亚单位疫苗；同时利用pull-down及质谱技术研究布菌优势抗原表位分子在动物巨噬细胞中可能存在的信号通路。此外，克隆布菌具有TIR结构域的蛋白质基因，利用双荧光素酶实验在果蝇S2细胞及人T293细胞中研究其对Toll样受体信号途径的阻断作用，并利用pull-down及Co-IP技术研究布菌具有TIR结构域蛋白与MyD88分子的结合作用，为新型疫苗的研制提供靶位点。

布鲁氏菌病是由布鲁氏菌引起的可在人、畜之间传播的二类传染病，至今没有可供人使用的布病疫苗，研制有效的动物及人用布病疫苗的需求十分迫切。本项目以布鲁氏菌BLS分子自身聚合作用作为立足点，通过蛋白质抗原与BLS分子的融合表达、亲和纯化、抗体制备等一系列研究内容，筛选得到omp10、omp19、omp22、omp25、omp28、omp31六个优势抗原表位基因，可以作为核酸亚单位疫苗、蛋白质亚单位疫苗的候选基因。同时，利用蛋白质融合表达技术，研究了布鲁氏菌BLS分子针对Omp31、L7/L12分子的免疫刺激提呈作用，结果显示BLS分子可以增强布鲁氏菌Omp31、L7/L12蛋白质抗原的免疫刺激效果，为BLS分子免疫刺激增强作用的实际利用奠定了理论基础。此外，利用pull-down技术研究了布鲁氏菌具有TIR结构域蛋白与MyD88分子的互作，结果显示布鲁氏菌具有TIR结构域蛋白与Toll信号途径胞内关键配体MyD88分子之间存在互作，初步探索了布鲁氏菌进入人体之后可以及时逃避宿主免疫监视作用的分子机制，为新型人用疫苗的研制提供了靶位点。