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研究生:許弘明
研究生(外文):Hong-Ming Hsu
論文名稱:陰道滴蟲tvMyb家族蛋白質生化特性分析
論文名稱(外文):Biochemical Characterization of tvMyb family proteins in Trichomonas vaginalis
指導教授:翁秀貞
指導教授(外文):Shiou-Jeng Ong
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:60
中文關鍵詞:生長轉錄因子陰道滴蟲Myb蛋白質
外文關鍵詞:Trichomonas vaginalisgirontranscription factor
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陰道滴蟲ap65-1啟動子上有兩種不同的Myb蛋白質辨認序列(Myb recognition element,MRE),分別為MRE-1/MRE-2r(TAACGATA)及MRE-2f(TATCGT)序列。前人從cDNA library篩選出一系列可能與MRE序列結合的tvMybs家族,命名為tvMyb1、tvMyb2、tvMyb3及tvMyb4。本研究以西方轉漬法證明,tvMyb1的表現量以及在細胞中的分佈不受鐵離子或生長影響;tvMyb2隨蟲體生長可能發生蛋白質修飾;tvMyb3蛋白質的表現量隨著生長時間先上升而後下降;tvMyb4早期同時分佈於細胞核與細胞質中,晚期則只存在細胞質內。進而以重組tvMyb蛋白質(rMyb),進行電泳遲滯分析,以確認rMyb與ap65-1啟動子上MRE序列的結合情形。四個rMybs蛋白質對不同MRE有其序列專一性。rMyb1結合包含MRE-1/MRE-2r之A-AACGAT序列以及MRE-2f內之ATCG序列。rMyb2結合MRE-2r內之CGATA序列以及包含MRE-2f之TATCGTC序列。rMyb3及rMyb4均結合在MRE-1。進而利用一系列刪短蛋白質,進行tvMyb的DNA結合弁鈰洃尷R;發現rMyb1之R2R3區域對MRE-1/MRE-2r及MRE-2f序列有相同的結合能力,其C端正向調控R2R3區域結合MRE-1/MRE-2r序列之能力,而負向調控R2R3區域結合MRE-2f序列之能力。rMyb1 N端負向調控R2R3區域結合兩種MRE序列之能力。rMyb3鄰近R2R3(53-150)之C端區域151-160,則正向調控rMyb3之DNA結合能力。由於各tvMybs的表現狀況與存在於細胞核內的時機各有不同,而且對MRE序列的結合強度不同,各tvMybs蛋白質和ap65-1啟動子結合時,可能有一種互相競爭的關係,藉此調控啟動子的活性。

The ap65-1 promoter in Trichomonas vaginalis is known to contain two Myb recognition elements 【MREs, MRE-1/MRE-2r(TAACGATA)and MRE-2f(TATCGT)】. Four Myb-like proteins (tvMybs), referred to as tvMyb1, tvMyb2, tvMyb3 and tvMyb4, which may interact with these MREs, were previously cloned from a T. vaginalis cDNA expression library. In this thesis, Western blotting and cellular fractionation experiments were employed to show that expression and subcellular localization of tvMyb1 are iron- and growth-independent, and tvMyb2 may be processed by post-translation modification upon growth. Expression of tvMyb3 was shown to be growth-dependent. tvMyb4 was shown to localize in nucleus and cytoplasm simultaneously at early stage, but only in cytoplasm at late stage. Electrophoretic mobility shift assay(EMSA)was then performed to study DNA binding specificity of recombinant Myb proteins (rMyb). rMyb1 was shown to interact with A-AACGAT (or ATCGAA-A in reverse) spanning MRE-1/MRE-2r, and ATCG in MRE-2f. rMyb2 was shown to recognize the sequence CGATA (or TATCG in reverse) in MRE-1/MRE-2r, and TATCGTC in MRE-2f. rMyb3 and rMyb4 were both found to interact with TAACGA in MRE-1. To analyze DNA binding domain and potential regulatory domains of tvMyb, a series of deleted rMyb1 were analyzd by EMSA. The data showed that the R2R3 domain of rMyb1 binds MRE-1/MRE-2r and MRE-2f equally, and C terminus positively regulates binding activity of R2R3 domain to bind MRE-1/MRE-2r but negatively regulates its R2R3 domain to bind MRE-2f. The N terminal of rMyb1 was shown to negatively regulate its R2R3 domain. Furthermore, the C terminal 151-160 amino acids closed to the R2R3 domain of rMyb3 were shown to positively regulate DNA binding ability of rMyb3. In summary, tvMybs probably use different expression profile and diverse binding specificity to compete with each other in regulating activity of the ap65-1 promoter.


縮寫表……………………………..……………….…………1
英文摘要………………………………….………..…………2
中文摘要………………………………….………..…………3
前言………………………………………………..……….…4
陰道滴蟲簡介………………………………………...……..4
陰道滴蟲基因轉錄……………………………….…..……..4
鐵離子與ap65-1基因啟動子…………….…………...…….5
Myb蛋白質家族……………………………………….....….6
研究目的…………………………………………..….……..8
材料與方法………………………………………..….………9
陰道滴蟲細胞株………………..……………..…………….9
蛋白質聚丙烯醯胺膠體電泳……….………..……………..9
西方轉漬(Western Blot)………………….……..………9
6×His-tag rMyb2重組蛋白質表現載體………...…………10
大量表現6×His-tag rMyb重組蛋白質…………..………..10
包涵體rMyb1蛋白質的重新折疊………………....……….11
6×His-tag rMyb蛋白質純化…………………….…….…..12
抗血清製備………………………………………..….…….12
抗體純化 …………………………………………...……..13
陰道滴蟲total lysate之收集……………….…..……….14
細胞核蛋白與細胞質蛋白萃取………………….…....….14
放射性寡聚核苷酸探針標定……………………...….……15
電泳遲滯分析……………………………….……..….…..15
結果……………………………..……………....……..…17
rMyb1重組蛋白質誘導表現及純化…………..….…..…..17
rMyb2重組蛋白質誘導表現及純化…………….....….….17
分析anti-Myb1、anti-Myb2抗體效價…………....……..18
鐵或生長對Myb蛋白質表現之影響…………..…....…….18
rMyb蛋白質之DNA結合特性…………………..…....…...19
rMyb1 DNA結合功能區分析…………………......…….…20
rMyb3 DNA結合功能區分析………………..…....…….…21
討論………………………………………….....….….….23
rMyb重組蛋白之純化與表現………………….…....….…23
鐵或生長對Myb蛋白質表現之影響………….......……..23
rMyb DNA結合特性……………………………........……25
rMyb蛋白質DNA結合功能區分析…………….......….….27
附圖..............................................29
參考文獻…………………………………………...….……51
附錄…………………………………………………...…….56

附圖目錄

圖一、ap65-1啟動子結構……………………………………29
圖二、pET30/myb2重組蛋白質表現載體…………….…….31
圖三、rMyb1重組蛋白質表現及純化………………….……33
圖四、rMyb2重組蛋白質表現及純化…………………….…35
圖五、血清效價分析…………………………………….….37
圖六、鐵濃度及生長時間對Myb蛋白質表現之影響……...39
圖七、鐵濃度及生長時間對Myb蛋白質在細胞內分布之影響.....41
圖八、rMyb蛋白質之DNA結合特性分析………………....……...43
圖九、rMyb1 DNA結合功能區之分析………………….…..……..45
圖十、rMyb3 DNA結合功能區之分析………………….……..……47
圖十一、rMyb3 DNA結合功能區分析………………….…..………49



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