布鲁氏菌疫苗靶标的筛选与验证

项目名称： 布鲁氏菌疫苗靶标的筛选与验证

项目编号： No.31070814

项目类型： 面上项目

立项/批准年度： 2011

项目学科： 医药、卫生

项目作者： 李蓓

作者单位： 湖北医药学院

项目金额： 8万元

中文摘要： 布鲁氏病是由布鲁氏菌引起的能够在人和主要经济动物牛、羊、猪中传播的一类人兽共患病，安全有效的疫苗接种是治疗和预防布鲁氏病的最有效手段。本课题从布鲁氏菌全基因组序列入手，根据反相疫苗学的原理，通过生物信息学进行分析，挑选出与细菌毒力、黏附、侵袭等相关的基因，利用Gateway克隆技术对挑选出的基因在体外大规模克隆、表达，获得90个能够满足蛋白芯片和Elispot实验要求的蛋白。然后结合蛋白芯片抗体谱技术，分别与感染布鲁氏菌人血清或布鲁氏菌疫苗株免疫鼠血清反应，检测布鲁氏菌感染后宿主产生体液免疫反应的情况。发现了3个新的能够刺激机体产生体液免疫的蛋白质。Elispot 检测了14个重组蛋白纯化产物体外刺激布鲁氏菌活疫苗株免疫小鼠脾细胞分泌IFN-γ#30340;细胞数。2 个蛋白能够明显的刺激布鲁氏菌活疫苗株免疫鼠脾细胞分泌IFN-γ12290;通过本课题了解布鲁氏菌活疫苗株在机体内产生免疫保护作用的机制，大规模鉴定布鲁氏菌中具有B细胞免疫原性和T细胞免疫原性的蛋白质，确定能用于下游疫苗开发的候选靶标分子。

中文关键词： 布鲁氏菌；疫苗；体液免疫；细胞免疫

英文摘要： Brucellosis is a worldwide zoonosis which caused by Brucella and also can infect human. Safe and validated vaccine is the most efficacious method to prevent and treat brucellosis. This project based on the genome of brucella to identify genes potentially ecoding surface-exposed proteins which associated with virulence,adherence and invasion of bacterium through computer analysis. All selected genes were colned into the expressed plasmids and expressed in E. coliBL21(DE3). The fusion recombinant proteins were high-throughput purified by affinity chromatography.Then the humoral and cellular immune responses of these proteins were evaluated by protein microarray and elispot technology. The antibody profile of patients and immunized mice were screened by protein microarray and 3 new antigens whose antibody can detected in all of them. The ability to stimulate cellular immune responses of 14 proteins were evaluated and found 2 new proteins.These proteins will be the targets for the next vaccine development of Brucella. Throught the study of this project will allow us to gain insights into brucellosis pathogenesis and discover more efficacious countermearsures.

英文关键词： Brucella;vaccine;humoral immunity;cellular immunity