本文關(guān)鍵詞： 日本腦炎病毒 非洲綠猴腎細胞(Vero細胞) 細胞膜蛋白 熱休克蛋白90β　出處：《第四軍醫(yī)大學》2010年碩士論文　論文類型：學位論文

【摘要】：日本腦炎病毒(Japanese encephalitis virus,JEV)屬于黃病毒科黃病毒屬,是具有包膜的單股正鏈RNA病毒,可引起急性中樞神經(jīng)系統(tǒng)感染,即日本腦炎(Japanese encephalitis,JE),重癥病死率可達5%~10%,幸存者后遺癥也在15%左右,嚴重威脅人類生命健康。近年來,JE的流行地域有不斷擴大的趨勢,已成為熱帶、亞熱帶地區(qū)非常嚴重的公共衛(wèi)生問題,更可作為潛在的生物戰(zhàn)劑使用,其重要性不容忽視。 盡管目前關(guān)于JE的預防、診斷、治療等都已經(jīng)有了較完善的方案,但其致病機理一直未明確,尤其是作為感染始動因素的JEV受體(JEVR)迄今尚未闡明。病毒與細胞膜受體的特異性結(jié)合是引發(fā)病毒感染宿主細胞的始動環(huán)節(jié),同時在決定病毒宿主特異性和細胞親嗜性方面起著重要作用。研究受體對于揭示病毒感染與免疫學機制,深入了解病毒與宿主細胞間相互關(guān)系,研制更有效的病毒疫苗,篩選診斷試劑和抗病毒藥物等都具有重要的意義。本研究課題旨在篩選非洲綠猴腎細胞(Vero細胞)膜上可與JEV結(jié)合的蛋白質(zhì)分子,為最終確定JEV在Vero細胞膜上的受體奠定基礎(chǔ)。 本研究采用溫和的非離子型去垢劑NP-40裂解提取Vero細胞膜蛋白,將未經(jīng)凍存的新鮮膜蛋白樣品與純化后的JEV及抗JEV單克隆抗體(mAb)2H4進行免疫共沉淀(co-immunoprecipitation, Co-IP),收集可與JEV結(jié)合的蛋白質(zhì)復合物,繼而經(jīng)SDS-PAGE,從上述復合物中分離出3條獨立的特異條帶,相對分子量分別為90 kDa、45 kDa和34 kDa。將這3條蛋白條帶從SDS-PAGE膠上切下約呈1 mm3小塊,進行質(zhì)譜(MS)分析,檢索人蛋白數(shù)據(jù)庫。結(jié)果提示,分子量約為90 kDa的蛋白為熱休克蛋白90β(HSP90β), 45 kDa和34 kDa均為未命名蛋白產(chǎn)物(unnamed protein product)。用抗HSP90βmAb進行流式細胞術(shù),所得結(jié)果用統(tǒng)計學方法均數(shù)±標準誤(x±s,n=4)表示,細胞本底的熒光百分比為1.64±0.06;JEV(MOI 1)與細胞的結(jié)合率用熒光百分比表示為89.9±0.72;加入抗HSP90β(1:50)后,JEV與細胞間的結(jié)合下降熒光百分比為79.91±1.9(P0.05);加入抗HSP90β的濃度提高為1:10時,JEV與細胞間的結(jié)合下降熒光百分比為68.28±2.79(P0.05)。證明抗HSP90βmAb可以明顯抑制JEV與Vero細胞結(jié)合。免疫熒光實驗(IFA)也得到同樣的結(jié)果。 上述結(jié)果初步表明Vero細胞膜表面存在的HSP90β可與JEV結(jié)合;HSP90β是否為Vero細胞膜上的JEVR尚需更多的生物學實驗予以驗證。
[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 鹵0.06 HSP90 / Moi _ 1) and the fluorescence percentage was 89.9 鹵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 鹵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 鹵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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373

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相關(guān)期刊論文 前1條

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