黑龙江立克次体表面蛋白与血管内皮细胞（宿主细胞）表面分子相互作用的研究

Rickettsia heilongjiangensis, pathogen of Far-Eastern spotted fever (FESF), was first isolated and identified in 1984 in Heilongjiang province in China. FESF has been recognized as an important emerging infectious disease in Northeast Asia. However,only a few of surface proteins of Rickettsia spp. have been demonstrated to be associated with their pathogenesis, involving the attachment and entry of rickettsiae to host cells and/or intracellular movement of rickettsiae. In our previous study, 25 surface-exposed proteins of R. heilongjiangensis were isolated.and identified using biotin-avidin technology combined with proteomics technology, and the recombinant surface-exposed proteins were prepared and used to fabricate a protein microarray. This project intends to screen potential ligand proteins from the identified surface-exposed proteins by probing the protein microarray with Cy5-labelled proteins of EAhy 926 cells (vascular endothelial cells, host cells). Ligand proteins adhering to the surface of host cells will be further confirmed with cell ELISA as well as an immunofluorescence assay. The receptor proteins on the surface of host cells will be identified by Pull-Down assay and protein microarray assay. The ligands and receptors will be co-localized by double-labelling immunofluorescence to confirm their interactions. Finally, the roles played by the ligand-receptor interaction during rickettsial attachment and entry to host cells will be further studied. The results obtained in this study will provide a novel knowledge to clarify pathogenic mechanism of R. heilongjiangensis and also provide candidate molecular targets for development of.vaccine and therapeutic drug against this pathogen.

黑龙江立克次体是我国首先发现的新斑点热病原体，其所致远东斑点热为东北亚地区一种重要的新发传染病。对立克次体分子致病机制的研究目前仅仅局限于几个已知的表面蛋白上。立克次体表面配体蛋白与宿主细胞受体蛋白相互作用在立克次体粘附和入侵宿主细胞、立克次体在宿主细胞内运动以及细胞间传播过程中具有重要作用。本课题拟利用前期工作中建立的黑龙江立克次体的表面蛋白芯片，筛选其中可能与宿主细胞（血管内皮细胞-EAhy 926细胞系）相互作用的蛋白，经细胞ELISA及免疫荧光技术进一步筛选其中能与宿主细胞表面蛋白相互作用的配体蛋白，利用Pull-Down及蛋白芯片技术鉴定宿主细胞上与其相配的受体蛋白，采用荧光共定位技术确定配体-受体相互作用，并深入分析其相互作用在黑龙江立克次体黏附和入侵宿主细胞过程中的作用机制。研究成果将为进一步阐明立克次体的致病机制提供理论依据，为斑点热的预防疫苗和治疗药物的研发提供候选靶标。

本研究基于我们前期建立的蛋白芯片平台，通过表面蛋白芯片与Cy5标记的EAhy 926细胞蛋白反应，对黑龙江立克次体表面配体进行了初步筛选，获得了包括OmpA-2、OmpB-2、Adr1、Adr2、YbgF在内的13种可能的配体蛋白，之后通过细胞ELISA进一步筛选，发现包括Adr1，Adr2，YbgF在内的7种蛋白均能黏附于宿主细胞表面，除Adr1及Adr2外，其余均为新发现的立克次体配体蛋白。这些蛋白可能在立克次体黏附和入侵宿主细胞、立克次体在宿主细胞内运动及细胞间传播过程中发挥重要作用，值得进一步研究，尤其是新发现的YbgF蛋白，可能在立克次体入侵宿主细胞中发挥重要作用。. 使用纯化的YbgF重组蛋白与人蛋白芯片HuProt反应，发现有808个宿主细胞蛋白具有与YbgF相互作用的倾向，然而我们筛选其中两个反应信号值高的Krtap-10和RTN4蛋白进行原核表达，成功表达和纯化了RTN4蛋白，然而在Pull-Down实验中并未发现其与YbgF蛋白相互作用，可能因为原核表达未成功实现RTN4蛋白的自然构象折叠。之后对YbgF蛋白与宿主细胞裂解液进行了Pull-Down实验，结果条带中未检测到目的蛋白，可能宿主细胞裂解液中蛋白丰度较低，难以实现检测。免疫荧光实验发现YbgF可以粘附于宿主细胞表面，血清中和实验确认YbgF蛋白具有阻止病原体入侵宿主细胞的作用，其受体蛋白尚需进一步确认。研究结果对阐明斑点热立克次体入侵宿主细胞机制有一定价值和意义。. 本研究共发表SCI论文4篇，培养研究生2名。