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研究生:陳錫銘
研究生(外文):Hsi-Ming Chen
論文名稱:HIV-1TAT重組蛋白傳導入哺乳動物細胞之研究
論文名稱(外文):The investigation on the transduction of HIV-1 TAT recombinant proteins into mammalian cells
指導教授:邱紫文邱紫文引用關係
指導教授(外文):Tzyy-Wen Chiou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:細胞穿透性胜�L蛋白質傳導HIV-1 TAT蛋白人類Aurora A蛋白
外文關鍵詞:cell-penetrating peptidesprotein transductionHIV-1 TAT proteinhuman Aurora A protein
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哺乳動物的細胞膜是大部分生物分子 (蛋白質、胜�L)進入細胞內的主要障礙。最近報告指出一些短胜�L序列具有穿越細胞脂質雙層膜的能力,稱之為細胞穿透性胜�L (cell-penetrating peptides;CPPs)。人類免疫缺陷病毒-1型 (HIV-1)的轉譯活化蛋白TAT,由研究報告得知TAT蛋白的鹼性胜�L傳導區 (PTD),是一段僅有11個胺基酸的短胜�L序列 (YGRKKRRQRRR),可穿透細胞生物膜。PTD的特性是帶有多個正電性精胺酸,可能和帶負電性脂質細胞膜的結合很重要。另外以化學鍵結或融合蛋白等方式,TAT-PTD已成功載運許多外源性蛋白進入多種細胞裡,包括體外培養細胞和老鼠活體器官組織細胞。

本實驗為了研究TAT重組蛋白的蛋白質傳導能力,以及外加中心體Aurora-A蛋白過量表現,對於人類子宮頸腫瘤 (HeLa)細胞的作用。我們使用基因重組和蛋白質的純化技術,並利用大腸桿菌表現系統,生產出四種TAT融合蛋白。TAT-HA-GFP蛋白以及TAT-HA-Aurora-A-GFP蛋白,經由蛋白質傳導培養30分鐘後,藉由帶有綠色螢光蛋白 (GFP)的特性,以螢光顯微鏡觀察TAT融合蛋白的傳導行為。

結果發現TAT-HA-GFP (36 kDa)蛋白能順利進入HeLa細胞的細胞質並堆積在細胞核內,進一步由共軛焦螢光顯微鏡亦確認是表現在細胞內。此結果支持TAT蛋白轉運載體系統能順利穿越細胞質膜並進入到細胞內。另外包含野生型(Wt.)、增活性型(T288D)、去活性型(K162M)之三種TAT-HA-Aurora-A-GFP (82 kDa)蛋白,使用共軛焦螢光顯微鏡分析顯示未能順利進入HeLa細胞,推論可能與Aurora-A-GFP大分子,在空間構形上包埋住N端的TAT-PTD位置,干擾了TAT-PTD的蛋白質傳導能力。

總言之,利用合成或基因重組等方法,設計含有傳導能力的細胞穿透性胜�L,與藥用性分子或細胞內作用蛋白結合,應可成為輔助醫學臨床治療或細胞蛋白體研究的有利工具。
The major obstacle for transducing most proteins and peptides into mammalian cells is to cross their membranes. Recently, reports have shown that some short peptides, such as cell-penetrating peptides (CPPs), are able to transverse the cellular lipid bilayers. The basic peptide transduction domain (TAT-PTD) containing only 11 amino acid residues (YGRKKRRQRRR) is derived from human immunodeficiency virus type-1 (HIV-1) transcriptional activator TAT, and is characterized by having a high content of positively charged arginine residues, which might be important for the contact with negatively charged cellular membrane lipids. TAT-PTD fused with heterologous proteins or peptides cross the cell biologic membranes efficiently. It has been reported that TAT-PTD can carry the cargos and delivered themselves into the cells not only ex-vivo cultured cells but also in vivo mouse living organs and tissues.

In order to study the TAT recombinant protein transduction permeability and the effect caused by overexpression of heterologous centrosomal Aurora-A proteins in HeLa cells, we expressed and purified four kinds of TAT recombinant fusion proteins by utilizing gene recombinant method and the denatured protein purification protocol in E. coli protein expression system. After protein transduction and for 30 minutes, we use fluorescence microscope to analysis the behavior of TAT fusion proteins, coupling with the reporter green fluorescent protein (GFP).

The results showed that TAT-HA-GFP protein could be transduced into the cytoplasm and accumulate in the cell nucleus. Furthermore, using laser-scanning confocal fluorescence microscopy, we confirmed the intracellular protein expression. It supports that TAT protein delivery system can cross the cell cytoplasm membrane successfully. In addition, we examined the TAT-HA-Aurora-A-GFP fusion proteins including wild-type, constitutive active form (T288) and inactive form (K162M) by confocal laser-scanning microscopy. Our results showed that none of the TAT-HA-Aurora-A-GFP recombinant proteins could successfully enter into HeLa cells. It was speculated that TAT-PTD site might be embedded by Aurora-A-GFP huge molecules in a special structural conformation, and that might hinder the TAT-PTD transductive ability.

In summary, via applying the chemical-link or recombinant proteins containing the cell-penetrating peptides that combine with the interesting drug molecules or intracellular interaction proteins, which could become one of the useful tools to assist clinical therapy development and cellular proteomic study.
第一章 緒論
1-1 HIV-1 TAT蛋白與蛋白質傳導 ……………………1
1-1-1 細胞穿透性胜�L介紹 …………………………1
1-1-2 HIV-1 TAT蛋白質的特性 ………………………5
1-1-3 HIV-1 TAT胜�L的結構與傳導現象 ……………8
1-2 中心體蛋白Aurora-A激�磥雯苤K…………………13
1-2-1 中心體功能介紹 ………………………………13
1-2-2 Aurora激�磥雯� ………………………………15
1-2-3 Aurora-A與腫瘤形成關係 ……………………18
1-3 研究目的 …………………………………………19

第二章 實驗材料與方法
2-1 實驗材料 …………………………………………20
2-2 TAT-Aurora-A-GFP質體的建構 …………………20
2-2-1 引子 (primer)的設計 ……………………20
2-2-2 PCR增殖Aurora-A基因 ……………………21
2-2-3 由洋菜膠萃取DNA …………………………21
2-2-4 DNA接合作用 (DNA Ligation) …………22
2-2-5 PCR 點突變 (PCR point mutation) ……22
2-2-6 轉型作用 (Transformation) …………22
2-2-7 質體DNA的小量製備 ………………………23
2-2-8 DNA定序 ……………………………………24
2-3 TAT融合蛋白之製備與分離 ……………………24
2-3-1 以大腸桿菌表現融合蛋白…………………24
2-3-2 超音波震盪破碎菌體 ……………………25
2-3-3 親和性管柱層析分離蛋白質………………26
2-3-4 SDS–PAGE蛋白質電泳 ……………………26
2-3-5 利用Slide-A膜快速透析TAT融合蛋白質…27
2-3-6 以Heparin層析管柱去除尿素 ……………28
2-3-7 以PD-10去鹽管柱置換溶液 ……………28
2-4 西方點墨法 (Western Blotting) ……………29
2-5 蛋白質傳導與螢光顯微鏡分析 …………………30
2-6 共軛焦螢光顯微鏡分析 …………………………31

第三章 結果 …………………………………………32
3-1 TAT融合蛋白質的純化和表現 …………………32
3-1-1 TAT-HA-GFP蛋白質生產 ………………………32
3-1-2 建構pTAT-HA-Aurora-A-GFP質體DNA…………36
3-1-3 TAT-HA-Aurora-A-GFP重組蛋白的表現………36
3-2 西方點墨法 (Western Blotting) ……………47
3-3 TAT-HA-GFP蛋白質傳導與螢光顯微鏡分析 ……47
3-4 TAT-HA-GFP蛋白共軛焦螢光顯微鏡分析 ………49
3-5 TAT-HA-Aurora-A-GFP蛋白螢光顯微鏡分析……52
3-6 TAT-HA-Aurora-A-GFP蛋白共軛焦螢光顯微鏡分析 …52

第四章 討論 ………………………………………56

第五章 參考文獻 ……………………………………62
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