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系統識別號 U0026-0812200911173254
論文名稱(中文) 登革病毒非結構性蛋白1與mucin-like 蛋白質的序列相似性: 抗登革病毒非結構性蛋白1抗體 交叉反應性之研究
論文名稱(英文) Dengue virus nonstructural protein 1 (NS1) shows sequence homology with mucin-like proteins: study on the cross-reactivity of anti-NS1 antibodies
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 92
學期 2
出版年 93
研究生(中文) 萬書彣
研究生(英文) Shu-Wen Wan
電子信箱 iamwanwan.tw@yahoo.com.tw
學號 s4691110
學位類別 碩士
語文別 中文
論文頁數 75頁
口試委員 口試委員-莊偉哲
口試委員-黃智雄
口試委員-黎煥耀
指導教授-林以行
中文關鍵字 登革病毒 
英文關鍵字 dengue 
學科別分類
中文摘要   登革病毒 (dengue virus; DV) 感染的病患會呈現不同嚴重程度的症狀,由輕微可自行痊癒的登革熱 (dengue fever; DF),到可能致死的登革出血熱 (dengue hemorrhagic fever; DHF) 或休克症狀 (dengue shock syndrome; DSS)。DHF/DSS的主要臨床病徵為血管病變滲漏、血小板減少症和出血,但是主要機制至今尚未清楚。實驗室之前的實驗結果顯示,不管是來自登革病患血清,還是來自利用登革病毒非結構性蛋白1 (dengue virus nonstructural protein 1;DV NS1) 多次免疫小鼠的血清,血清中的抗登革病毒非結構性蛋白1抗體(anti-DV NS1 antibodies) 皆能交叉反應於人類的內皮細胞,並且造成細胞的死亡。本篇論文主要是探討內皮細胞和NS1蛋白間是否有相似的抗原決定位 (epitope)。利用GCG database比對,發現在DV NS1胺基酸序列312-331與人類mucin 5AC和mucin 5B具有序列相似性。而mucin 5AC和mucin 5B普遍地表現在許多細胞的表面,特別是上皮細胞,我們實驗也證明人類內皮細胞也會表現mucin 5AC/5B。並且抗mucin 5AC及抗mucin 5B抗體也能夠交叉反應於重組DV NS1蛋白及NS1蛋白C端胺基酸序列312-331的胜肽 (C20),但無法交叉反應於N端胺基酸序列1-15的胜肽 (N15)。此外,anti-DV NS抗體和anti-mucin 5AC/5B抗體結合於HMEC-1或是A549細胞的能力分別都會被anti-mucin 5AC/5B抗體和anti-DV NS抗體部分競爭抑制。在登革病人方面,血清中anti-C20 and anti-M20 IgM的效價與結合於HMEC-1和A549細胞具有正相關性。若利用胜肽C20和M20 (在mucin 5AC/5B序列中與C20類似的片段) 免疫小鼠所產生的抗體皆可結合於HMEC-1細胞。利用基因重組蛋白技術去除DV NS1胺基酸序列278-352的truncated DV NS1競爭抑制anti-DV NS1抗體結合於HMEC-1細胞的能力與DV NS1的程度大致相同,但是C20、M20便無法抑制。因此,HMEC-1細胞表面的mucin-like domain與DV NS1具有分子相似性,但是這個DV NS1上的抗原決定位是否扮演一角色,仍有待進一步釐清。


英文摘要   Infection with dengue virus (DV) causes diseases ranging widely in severity, from self-limited dengue fever (DF) to life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Vascular leakage, thrombocytopenia, and bleeding are the clinical manifestations associated with dengue infection, yet the mechanisms remain unclear. Previous studies in our laboratory showed that anti-DV nonstructural protein 1 (NS1) antibodies (Abs) from both dengue patient sera and DV NS1 hyperimmunized mouse sera cross-reacted with human endothelial cells and induced damage. In this study, the epitopes shared between cell surface molecules and NS1 are identified. The epitope of C-terminal DV NS1 amino acid (a.a.) 312-331 shows homology with human mucin 5AC and mucin 5B analyzed by GCG database. Human mucin 5AC and 5B are commonly expressed on the surface of many cells, especially in epithelial cells. We have confirmed that mucin 5AC and 5B are also expressed in human endothelial cells. Results showed that anti-mucin 5AC and 5B Abs were able to bind recombinant DV NS1 and C-terminal peptides (C20, a.a. 312-331) but not N-terminal peptides (N15, a.a. 1-15) by ELISA. The competitive binding assay showed that the binding activities of anti-DV NS1 Abs and anti-mucin 5AC/5B Abs to HMEC-1 or A549 cells were partially inhibited by preabsorption with anti-mucin 5AC/5B and anti-DV NS1 Abs, respectively. The anti-C20 and anti-M20 IgM titers of dengue patient sera are positively correlated with the binding activity of dengue patient sera IgM with HMEC-1 and A549 cells. The anti-C20 Abs and anti-M20 Abs (M20 is homologous with C20 and is present on muicn 5AC/5B) could also cross-react with HMEC-1 cells. Recombinant DV NS1 protein and truncated DV NS1 (DV NS1 a.a. 278-352 deletion) can both inhibit the cross-reactivity of anti-DV NS1 with HMEC-1 cells, but C20, M20 peptides can not. Our data therefore demonstrate a potential epitope of mucin-like domain which shows molecular mimicry with DV NS1, but whether this epitope may play a role in disease pathogenesis remains to be clarified.


論文目次 中文摘要
英文摘要
致謝
目錄
表目錄
圖目錄
符號及縮寫索引表

緒論
特定目標與實驗設計
材料與方法
A. 材料
A-1 實驗動物
A-2 重組蛋白與胜肽合成
A-3 細胞株
A-4 病患血清
A-5 試劑藥品
A-6 抗體
A-7 耗材
A-8 儀器
B. 方法
B-1 NS1重組蛋白之製備
B-2 SDS-PAGE膠體電泳
B-3 蛋白質定量
B-4 NS1重組蛋白免疫小鼠及血清的製備
B-5抗體的製備與特性分析
B-6 細胞的分離與培養
B-7 細胞與抗體的作用
B-8 抗體與重組蛋白DV NS1及胜肽間的交叉反應
B-9 抗體競爭結合於細胞的能力
B-10 Truncated DV NS1蛋白的製備
結果
一、 探討anti-DV NS1抗體與anti-mucin 5AC/5B抗體結合到內皮細胞的關係
二、 利用合成胜肽免疫小鼠後所產生的抗體,探討其抗體與內皮細胞間的關係
三、 將DV NS1蛋白胺基酸序列之C端可能的抗原決定位去除後,分析其免疫抗原性
討論
自體免疫的理論基礎
自體抗體的病理效應
登革病毒感染引發自體免疫
DV NS1的C端胺基酸序列與anti-DV NS1抗體結合於
HMEC-1的關連性
日本腦炎病毒感染與自體免疫發生的關連性
研發登革疫苗的考量
參考文獻


附錄: 試劑配方
作者簡歷
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