人血管内皮细胞登革病毒候选受体- - 55 kDa蛋白的鉴定

项目名称： 人血管内皮细胞登革病毒候选受体- - 55 kDa蛋白的鉴定

项目编号： No.31200133

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 微生物学、植物学

项目作者： 杨杰

作者单位： 中国人民解放军第三军医大学

项目金额： 22万元

中文摘要： 登革病毒(dengue virus, DENV)是引起登革热、登革出血热/登革休克综合征(DF, DHF/DSS)的元凶，广泛流行于热带和亚热带地区，严重危害人类健康，但目前对其所致疾病既无特异治疗手段也无有效疫苗预防。病毒与敏感细胞表面受体结合是发生病毒感染的第一步。研究表明，人血管内皮细胞（VEC）是登革感染的重要靶细胞，但该细胞上DENV受体迄今尚未阐明。本研究在前期用改良的病毒铺覆蛋白印迹技术（VOPBA）筛选出人VEC上新的DENV受体候选分子（55 kDa蛋白）基础上，拟采用2D电泳进一步分离候选蛋白，经N-端测序和质谱分析确定其编码序列，再进行候选蛋白的表达和抗体制备，Co-IP和受体重塑实验鉴定这一新发现的DENV受体，最后通过Pull-down实验对新鉴定受体的功能区进行定位。为深入认识登革致病机理、研发受体特异性阻断剂，进而控制DENV感染和流行奠定坚实基础。

中文关键词： 登革病毒；血管内皮细胞；包膜糖蛋白III区；波形蛋白；对接模型

英文摘要： Dengue disease that caused by dengue virus (DENV) is a steadily worsening health problem in the tropical and subtropical areas of the world. DENV can result in a spectrum of clinical symptoms from dengue fever to dengue hemorrhagic fever / dengue shock syndrome (DF, DHF / DSS)，and no therapy or effective vaccines are available at present. The binding of virus to a surface receptor of the sensitive cell is the first step for viral infection. Many researches show that human vascular endothelial cells (VECs) are the main target for DENV infection. However, the exact molecules that afford DENV entry into the VECs have not been clarified yet. This research is based on a newly identified DENV receptor candidate molecule (55 kDa protein) on VEC suffer, screened by a modified virus overlay protein blot assay (VOPBA). In this proposal,we plan to purify the candidate protein by two-dimensional electrophoresis, and determine its coding sequence by N-terminal sequencing and mass spectrometry. Then we prepare the candidate protein in large amounts through recombinant technology for generating antibody. After that, using Co-IP and the receptor remodeling experiment, we would like to confirm the newly discovered protein as a DENV receptor. Finally,the functional domains of this receptor will be determined through a pull-down

英文关键词： Dengue virus；VECs；EDIII；vimentin；docking modle