布鲁氏菌在宿主细胞内形成复制性菌泡是其在胞内生存和增殖的关键。研究发现布鲁氏菌能利用宿主细胞的小G蛋白Sar1形成复制性菌泡，且布鲁氏菌Ⅳ型分泌系统也能调节复制性菌泡的形成，但是Sar1和Ⅳ型分泌系统之间的关系目前尚不清楚。我们的预实验显示：Sar1能与效应蛋白相结合。据此，我们推测布鲁氏菌可能通过Ⅳ型分泌系统分泌的效应蛋白获取宿主细胞的Sar1从而调节复制性菌泡的形成。为了证实这一假说，本课题拟通过GST -Sar1融合蛋白沉降、二维电泳、质谱分析、酵母生长抑制实验及效应蛋白表达实验筛选出IV型分泌系统分泌且能与Sar1互作的效应蛋白；然后构建缺失效应蛋白的布鲁氏菌株；通过检测布鲁氏菌中期菌泡与溶酶体或内质网融合情况来探究效应蛋白在复制菌性泡形成中的作用。本课题的实施将为布鲁氏菌胞内生存机制研究提供新思路，同时，某种关键效应蛋白的发现可能为布鲁氏菌病的治疗提供潜在靶点。

The formation of replicative Brucella-containing vacuole (rBCV) is crucial for survival and replication of Brucella. The small GTPase Sar1 that ensures newly manufactured proteins in endoplasmic reticulum (ER) are packaged properly and addressed for correct delivery to other organelles. Brucella can coopt Sar1 for the formation of rBCV. However, the exact mechanism remains unknown. A mutant of VirB type IV secretion system, which is necessary for rBCV building, was unable to sustain interaction and fuse with ER, and was killed via eventual fusion with lysosomes. Moreover, our preliminary data showed that Sar1 can interact with secreted effector from Brucella. Based on the above observations, we propose whether Brucella secreted effector specially interacting with Sar1 mediates the formation of rBCV. To test the hypothesis, VirB type IV secretion system-dependent effectors are identified by GST-Sar1 pull-down, analysis of 2 dimensional (2D) gel electrophoresis and mass spectrometry, suppression assay of yeast growth and a functional assay of type IV secretion system. According to the characteristics of the effector, an in-frame deletion mutant will be constructed for the effector coding sequence (CDS) in B.abortus 2308 strain. The following experiments will focus on the molecular mechanism of rBCV formation and Brucella survival by the effector: (i) the roles of effector in Sar1 recruitment to rBCV; (ii) the roles of effector in fusion with ER; (iii) the roles of effector in avoiding fusion with lysosome; (iv) the roles of effector in Brucella survival within macrophages. These studies will not only help elucidate how effector regulates the formation of rBCV and show a novel interpretation of Brucella survival, but will potentially give an insight in how to manipulate the bacterial effector to achieve more efficient elimination of intracellular Brucella. This is a topic of significance in the development of drugs to prevent and treat Brucellosis.