牛支原体分泌蛋白MbovP0725去磷酸化修饰调控机制研究

牛支原体是影响牛业健康发展的重要病原体，目前尚无高效疫苗和药物用于防治，其致病机制不清是缺少防控措施的根本原因。申请人所在课题组前期研究发现一种分泌性磷酸酶MbovP0725在牛支原体强、弱菌株中存在差异表达，可减弱牛支原体诱导宿主MAPKp38的磷酸化水平。本项目拟从牛支原体和宿主两个层面，深入探索该蛋白去磷酸化的调控作用。首先从蛋白水平确定其磷酸酶活性和底物特异性，分别构建点突变和基因片段缺失突变体确定活性位点和功能区，从随机肽库中确定宿主磷酸化保守基序；利用蛋白双向电泳及质谱技术筛选突变株、回补株、野生株差异磷酸化蛋白，PRM技术进行验证；然后从宿主角度，利用细胞转染、Co-IP、质谱、磷酸化抗体芯片等全面分析MbovP0725与宿主细胞的蛋白互作网络以及对牛支原体宿主细胞炎性反应的影响。研究结果将为阐明牛支原体致病因子和致病机制提供依据，为开发新型药物、疫苗、诊断试剂提供靶点。

Mycoplasma bovis is a prime pathogen affecting the healthy development of cattle industry. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The unclear pathogenesis is widely considered an underlying cause of lacking the effective application of control measures. Our previous research found that MbovP0725 encoded a haloacid dehalogenase has phosphatase enzyme activity involved in the secretion process of M.bovis and showed differential expressed between high and low virulence strains. Moreover, MbovP0725 could attenuate activation of host MPAK p38 pathway. This project is aimed to confirm its phosphatase activity and explore its regulatory effects on the pathogen and host. Firstly, we will plan to determine its enzyme activity and identify the specific substrate and phosphorylation site by means of site directed mutagenesis and fragment deletion mutagenesis. Then from a random peptide library, we will further identify the specific affinity peptides and analyze its consensus motif. The differential phosphorylated proteins from M.bovis wildtype strain, Mbov_0725 mutants and its complement strain will be identified by two-dimensional gel electrophoresis and mass spectrometry. The enriched proteins will be analyzed and selected for PRM target verification. Then after BoMac cell culture and transfection with endotoxin-free plasmid containing Mbov_0725, co-immunoprecipitation and mass spectrometry, phosphorylated antibody chip detection, we will to screen and identify the phosphorylation cascades targeted for MbovP0725 in key host signal pathway. And then we will determine its effect on the inflammatory response during M.bovis infected process. The results will provide a basis for clarifying the virulence factors and pathogenic mechanism of M.bovis, and provide targets for the development of new drugs, vaccines and diagnostic reagents.