灵长类动物细胞膜上日本脑炎病毒受体蛋白的鉴定

项目名称： 灵长类动物细胞膜上日本脑炎病毒受体蛋白的鉴定

项目编号： No.30872216

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 丁天兵

作者单位： 中国人民解放军第四军医大学

项目金额： 32万元

中文摘要： 大规模培养HeLa和Vero细胞，收获细胞，提取细胞膜蛋白组分，与JEV进行免疫共沉淀实验，再经SDS-PAGE，显示各有3条明显的特异条带，质谱分析显示均有热休克蛋白90β65288;HSP90β65289;，故选择HSP90β36827;行进一步鉴定。 ELISA实验证实HSP90β21487;与JEV体外结合，激光扫描共聚焦显微镜显示HSP90β19982;JEV共定位于细胞膜上，表明HSP90β22312;细胞膜上确有存在。免疫荧光和流式细胞术实验结果也显示JEV与HSP90β30340;结合可被抗HSP90β25239;体抑制，抑制率与抗体浓度成正比，显示出剂量依赖效应；抗HSP90β25239;体可推迟JEV感染HeLa和Vero细胞后细胞病变的出现，且与抗体浓度成正比。HSP90特异性抑制剂Geldanamycin对JEV感染无明显影响。因Geldanamycin不能抑制HSP90β34920;达，故设计、合成了4个针对HSP90β19981;同位点的小干扰RNA片段，目前正对HSP90β34920;达抑制效果进行评价，随后将观察其对JEV感染性的影响。 本研究结果显示HSP90β26159;灵长类动物细胞HeLa和Vero的JEV受体，为最终阐明JEV感染细胞的分子机制奠定扎实的实验基础。

中文关键词： 日本脑炎病毒；受体；HeLa细胞；Vero细胞；热休克蛋白90β

英文摘要： HeLa and Vero cells in large quantity were harvested, and membrane proteins were extracted, co-immunoprecipitated with JEV, and subjected to SDS-PAGE. Three specific bands displayed on gels were cut for mass spectrometry, and the results indicated that heat shock protein 90 βHSP90βboth appeared in HeLa and Vero cell samples. Hence, HSP90βas chosen for further identification. HSP90βas shown to be capable of binding JEV in vitro by ELISA. Confocal laser scan microscopy results demonstrated that HSP90βnd JEV co-located on cell membrane, indicating that the cytosolic HSP90βid rest on cell membrane. Immunofluorescent assay and flow cytometric results exhibited that the binding of HSP90βnd JEV was inhibited by specific anti-HSP90βntibodies, and the inhibition rates were proportionate with antibody concentrations in a manner of dose dependence. In addition, anti-HSP90βntibodies deferred the advent of cytopathic effects by JEV infection to HeLa and Vero cells, also revealing a dose-dependent effect. Geldanamycin, a specific HSP90 inhibitor, did not produce conspicuous inhibitory influence on JEV infectivity as Geldanamycin does not repress HSP90βxpression in cells. Thus, aiming to knock down HSP90βxpression, three different small interfering RNA fragments were designed, synthesized, and is evaluated underway, following the investigation of final impact on JEV infectivity under the condition of suppressed HSP90βxpression. Together, above research results indicated that HSP90βas a putative JEV receptor on primate HeLa and Vero cells.

英文关键词： Japanese encephalitis virus; receptor; HeLa cell; Vero cell; heat shock protein 90βHSP90β