Vero细胞膜上与日本脑炎病毒结合分子的初步筛选

发布时间：2018-03-01 15:26

本文关键词： 日本脑炎病毒非洲绿猴肾细胞(Vero细胞) 细胞膜蛋白热休克蛋白90β　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】：日本脑炎病毒(Japanese encephalitis virus,JEV)属于黄病毒科黄病毒属,是具有包膜的单股正链RNA病毒,可引起急性中枢神经系统感染,即日本脑炎(Japanese encephalitis,JE),重症病死率可达5%~10%,幸存者后遗症也在15%左右,严重威胁人类生命健康。近年来,JE的流行地域有不断扩大的趋势,已成为热带、亚热带地区非常严重的公共卫生问题,更可作为潜在的生物战剂使用,其重要性不容忽视。尽管目前关于JE的预防、诊断、治疗等都已经有了较完善的方案,但其致病机理一直未明确,尤其是作为感染始动因素的JEV受体(JEVR)迄今尚未阐明。病毒与细胞膜受体的特异性结合是引发病毒感染宿主细胞的始动环节,同时在决定病毒宿主特异性和细胞亲嗜性方面起着重要作用。研究受体对于揭示病毒感染与免疫学机制,深入了解病毒与宿主细胞间相互关系,研制更有效的病毒疫苗,筛选诊断试剂和抗病毒药物等都具有重要的意义。本研究课题旨在筛选非洲绿猴肾细胞(Vero细胞)膜上可与JEV结合的蛋白质分子,为最终确定JEV在Vero细胞膜上的受体奠定基础。本研究采用温和的非离子型去垢剂NP-40裂解提取Vero细胞膜蛋白,将未经冻存的新鲜膜蛋白样品与纯化后的JEV及抗JEV单克隆抗体(mAb)2H4进行免疫共沉淀(co-immunoprecipitation, Co-IP),收集可与JEV结合的蛋白质复合物,继而经SDS-PAGE,从上述复合物中分离出3条独立的特异条带,相对分子量分别为90 kDa、45 kDa和34 kDa。将这3条蛋白条带从SDS-PAGE胶上切下约呈1 mm3小块,进行质谱(MS)分析,检索人蛋白数据库。结果提示,分子量约为90 kDa的蛋白为热休克蛋白90β(HSP90β), 45 kDa和34 kDa均为未命名蛋白产物(unnamed protein product)。用抗HSP90βmAb进行流式细胞术,所得结果用统计学方法均数±标准误(x±s,n=4)表示,细胞本底的荧光百分比为1.64±0.06;JEV(MOI 1)与细胞的结合率用荧光百分比表示为89.9±0.72;加入抗HSP90β(1:50)后,JEV与细胞间的结合下降荧光百分比为79.91±1.9(P0.05);加入抗HSP90β的浓度提高为1:10时,JEV与细胞间的结合下降荧光百分比为68.28±2.79(P0.05)。证明抗HSP90βmAb可以明显抑制JEV与Vero细胞结合。免疫荧光实验(IFA)也得到同样的结果。上述结果初步表明Vero细胞膜表面存在的HSP90β可与JEV结合;HSP90β是否为Vero细胞膜上的JEVR尚需更多的生物学实验予以验证。
[Abstract]:Japanese encephalitis virus belongs to the family flaviridae. It is a single-stranded positive strand RNA virus with envelope. It can cause acute central nervous system infection, that is, Japanese encephalitis virus JEV. The severe death rate can reach 5% and the survivors' sequelae are about 15%. In recent years, the epidemic area of JE has become a very serious public health problem in the tropics and subtropics, which can be used as a potential biological warfare agent, and its importance can not be ignored. Although the prevention, diagnosis and treatment of je have been improved, the pathogenesis of je has not been clear. Especially, the JEV receptor (JEVR), which is the initiator of infection, has not been elucidated up to now. The specific binding between virus and cell membrane receptor is the initiation of viral infection in host cells. At the same time, it plays an important role in determining the host specificity and cell tropism of virus. The study of receptor is useful in revealing the mechanism of virus infection and immunology, understanding the relationship between virus and host cells, and developing a more effective virus vaccine. Screening of diagnostic reagents and antiviral drugs is of great significance. The aim of this study was to screen JEV binding protein molecules on the membrane of African green monkey kidney cells, so as to lay a foundation for the final identification of JEV receptor on Vero cell membrane. In this study, Vero membrane proteins were extracted by NP-40, a mild non-ionic descaling agent. Unfrozen fresh membrane protein samples and purified JEV and anti-mAb2H4 monoclonal antibody were immunoprecipitated co-immunoprecipitation (Co-IPP), and protein complexes that could bind to JEV were collected. Three independent specific bands were isolated from the above complexes by SDS-PAGE. The relative molecular weights were 90 kDa 45 kDa and 34 kDa respectively. The three protein bands were cut off from the SDS-PAGE gel to present about 1 mm3 fragment, and then analyzed by MS) to search the human protein database. The protein with molecular weight of about 90 kDa was heat shock protein 90 尾 -HSP90 尾, and 45 kDa and 34 kDa were unnamed protein products. Flow cytometry was performed with anti-#en4# 尾 mAb. The fluorescence percentage of the cell background was 1.64 卤0.06 HSP90 / Moi _ 1) and the fluorescence percentage was 89.9 卤0.72; after the addition of anti-JEV 尾 1: 50), the fluorescence percentage of the binding between the cell and the cell was 79.91 卤1.9 (P0.05); the concentration of anti-JEV 尾 was increased to the level of the intercellular between the HSP90 尾 and the cell at 1:10. The percentage of decreased binding fluorescence was 68.28 卤2.79 (P0.05). It was proved that anti-#en0# 尾 mAb could significantly inhibit the binding of JEV to Vero cells, and the same result was obtained by immunofluorescence assay (IFA). These results indicated that the existence of HSP90 尾 on the surface of Vero cell membrane and JEV binding to HSP90 尾 could be confirmed by more biological experiments.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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