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研究生:王重雅
研究生(外文):Chung-Ya Wang
論文名稱:埃及斑蚊體內含硫酯蛋白1(AaTEP1)基因之調控研究
論文名稱(外文):Regulation Study of a Thioester-containing Protein 1(AaTEP1)Gene from Aedes aegypti
指導教授:卓文隆
指導教授(外文):Wen-Long Cho
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:101
中文關鍵詞:埃及斑蚊含硫酯蛋白
外文關鍵詞:Aedes aegyptiTEP1
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含硫酯蛋白(thioester-containing proteins,TEPs)普遍存在於生物體中,在脊椎動物裡具代表性的便是補體因子C3/C4/C5以及蛋白酶抑制劑α2-macroglobulins;這類蛋白質具有辨識外來分子之功能,可標記外來物促進其吞噬作用或引起細胞溶解,在免疫反應上扮演了重要角色。而昆蟲同樣具有含硫酯蛋白,在果蠅體內已發現tep1可被細菌誘發並具有補體的功能,且可透過JAK-STAT路徑來調控;在瘧蚊體內的TEP1也同樣被發現可抵抗瘧原蟲的感染。本實驗室先前已成功地選殖出埃及斑蚊AaTEP1,對於其序列、結構和特性已有初步了解。本研究中發現埃及斑蚊給予革蘭氏陽性菌S. aureus刺激,體內AaTEP1 mRNA可大量表現。此外,為了深入探討AaTEP1和免疫調控路徑的關係,我們利用雙股RNA干擾法(dsRNAi)和抑制劑試驗(inhibitor assay),分析Toll路徑中Pelle基因、Imd路徑中的Relish2基因,以及p38 MAP kinase路徑和AaTEP1的調控關係;研究結果發現Aag2細胞株和埃及斑蚊AaTEP1皆可受到Relish2基因之調控,而和Toll與p38 MAP kinase調控路徑較無關係。進一步地從AaTEP1序列上分析啟動子附近可能的轉錄因子結合位(elements),將AaTEP1基因上游調控序列連結至螢光或冷光報導基因(fluorescent or luciferase reporter genes)後,利用系列刪除法分析其基因調控特性,結果顯示有一個Repressor結合位在基因的-1190至-1177區域可明顯影響啟動子表現能力,而Dorsal(-619至-606)和TATA結合位(-354至-338)則和細菌刺激後的誘發表現有明顯相關性。
Thioester-containing proteins(TEPs)act as potential recognition molecules in immune responses of vertebrates, such as complement factors C3/C4/C5 and α2-macroglobulins, which can label pathogens and promote phagocytosis or cell lysis. TEP is also an important component of the innate immune response in insects. It belongs to a hemocyte-specific acute phase glycoprotein, and is secreted into mosquito hemolymph and proteolytically processed shortly after septic injury. Aedes aegypti TEP1 (AaTEP1) has been cloned and sequenced in our laboratory. In this study, the expression patterns of AaTEP1 were investigated intensively in mosquitoes and mosquito cell lines. The mRNA of AaTEP1 was dramatically induced by gram positive bacteria Staphylococcus aureus (S. aureus) in mosquitoes. In order to study the regulation pathway of AaTEP1 involved in immunity, double stranded RNA interference (dsRNAi) was used to verify that control of AaTEP1 expression was mediated through Relish2 regulation pathway. In addition, the regulatory regions of AaTEP1 gene were isolated and characterized at the sequence level. A 1.7 kb fragment of AaTEP1 upstream region containing several binding elements were ligated to fluorescent and luciferase reporter genes, and the regulation of AaTEP1 gene was examined after different pathogen challenges. Serial deletions of the AaTEP1 promoter region were conducted for functional assay. It revealed that a Repressor binding site, a Dorsal binding site, and a TATA box were important for bacterial response regulation in AaTEP1 gene.
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縮寫表 ……………………………………………… ii
中文摘要 …………………………………………… iii
英文摘要 …………………………………………… V
目錄 ………………………………………………… Vii
壹、緒論…………………………………………… 1
貳、實驗材料與方法……………………………… 16
參、結果…………………………………………… 33
肆、討論…………………………………………… 44
伍、參考文獻……………………………………… 52
陸、圖……………………………………………… 56
柒、附錄…………………………………………… 86
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