贝氏柯克斯体为重要人兽共患病Q热的病原体，对人和动物有极高感染性。贝氏柯克斯体分泌至宿主胞质的效应蛋白是其胞内生存和致病的关键因子。目前已鉴定的贝氏柯克斯体效应蛋白中，绝大多数的生物学功能与宿主靶点仍未明确。因此，本研究首先构建含有Strep等标签的效应蛋白表达载体，并转染真核细胞系，观察效应蛋白的胞内定位及毒性等；然后采用亲和层析联合串联质谱技术鉴定宿主细胞内与效应蛋白相互作用的人靶蛋白，运用免疫共沉淀等技术验证它们的相互作用，绘制效应蛋白与人靶蛋白的互作网络；最后选取1～2对新鉴定的蛋白质互作对进行深入研究。构建相应的效应蛋白基因缺失和回补菌株，在细胞和动物模型上分析其毒力表型的变化；构建相应的人靶蛋白敲低或过表达细胞系和基因敲除小鼠，分析其对贝氏柯克斯体感染的影响。本项目将揭示1～2种效应蛋白与人靶蛋白的相互作用在贝氏柯克斯体致病中的作用及机制，为Q热的防治提供新靶标。

Coxiella burnetii is the causative agent of Q fever, which is highly infectious to both human and animals. Effector proteins of C. burnetii that delivered into the cytosol of host cells are determinants for its intracellular replication and pathogenicity. For the majority of the identified effectors, the functions and host targets are still uncharacterized. To uncover the underlying functions of the effector proteins, we will express effector-Strep fusion proteins in eukaryotic cells and analyze their subcellular location and cytotoxicity. Then we will apply a systematic affinity tag purification mass spectrometry（AP-MS）approach to identify the effector-human protein-protein interactions (PPIs). After that, we will apply Co-Immunoprecipitation and GST-Pull Down assay to confirm the PPIs in vivo and in vitro, establishing the PPIs network between the effector proteins and human target proteins. In addition, we will investigate the role of 1 or 2 of the identified effector-human PPIs involved in the pathogenicity of C. burnetii. For the selected effectors, we will create the targeted gene deletion and complementation of C. burnetii, and test their virulence phenotype in cellular and animal models. For the identified human target proteins, we will construct cell lines with targeted gene silenced or overexpressed and construct mice with targeted gene knockout, then investigate their relevance to the intracellular replication of C. burnetii. Our study will reveal the function and mechanisms of the effector-human PPIs in the pathogenicity of C. burnetii and provide new targets for the prevention and therapy of Q fever.