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研究生:林佳穎
研究生(外文):LIN, CHIA YING
論文名稱:研究含有Eps15同源結構域蛋白與液泡分選蛋白35之間的相互作用
論文名稱(外文):To investigate the interaction between Eps 15 homology domain-containing protein and vacuolar protein sorting 35
指導教授:黃憲斌
指導教授(外文):HUANG, HSIEN-BIN
口試委員:李沁呂明錡黃憲斌
口試委員(外文):LI, CHINLU, MING-CHIHUANG, HSIEN-BIN
口試日期:2022-07-28
學位類別:碩士
校院名稱:國立中正大學
系所名稱:生命科學系生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:64
中文關鍵詞:Eps15同源結構域蛋白液泡分選蛋白35phostensin結合模序運鐵蛋白EHD蛋白質胞吞運輸
外文關鍵詞:Eps 15 homology domain-containing proteinvacuolar protein sorting 35phostensinbinding motiftransferrinEHD proteinendocytic trafficking
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Eps 15同源結構域(Eps 15 homology domain, EHD)蛋白在胞吞運輸(endocytic trafficking)上扮演重要的角色。EHD蛋白質有四個可辨識區域,從N-端到C-端分別為N-端螺旋域、動力蛋白域(又稱G結合域)、中央捲曲旋轉域與C-端EH區域。液泡分選蛋白35 (Vacuolar protein sorting 35, Vps35)是逆向異構體中的一個次單元,此異構體在內體 (endosome) 分選多種貨物分子中扮演重要作用。
在之前的研究中,我們在phostensin (PTS)中發現了一個新的模序ILV(X)4(L/V)RLS,它與Eps 15同源結構域蛋白1 (EHD1)或EHD4的動力蛋白域結合。該模序殘基的定點突變將大幅減少PTS與EHD1或 EHD4的結合。有趣的是,液泡分選蛋白35(Vps35)也包含這個模序。我的研究是要探討是否Vps35這個模序也參與了與 EHD1或 EHD4的相互作用。

Eps 15 homology domain-containing (EHD) proteins play a key role in the endocytic trafficking. EHD proteins contain four recognizable domains from N-terminus to C-terminus:an N-terminal helical domain (NT), a dynamin-like domain (G domain), a central coiled-coil region and a EH domain. Vacuolar protein sorting 35 (Vps35) is a subunit of retromer complex that also plays important roles in endosomal sorting of a variety of cargo molecules.
In the previous study, we have identified a novel motif, ILV(X)4(L/V)RLS, in phostensin (PTS) that binds to the dynamin-like domain of Eps 15 homology domain-containing protein 1 (EHD1) or EHD4. Site-directed mutagenesis at the residues of this motif will significantly reduce PTS binding to either EHD1 or EHD4. Interestingly, vacuolar protein sorting 35 (Vps35) also contains this motif. In my study, I investigated whether this motif of Vps35 is also involved in interaction with either EHD1 or EHD4.

摘要 I
Abstract II
目錄 III
圖目錄 V
第一章 背景介紹 1
1.1囊泡運輸(Vesicular trafficking) 1
1.2胞吞作用(Endocytosis) 3
1.3胞吞循環途徑(Endocytic recycling) 4
1.4 Eps 15 homology domain (EHD) containing protein 5
1.5 Vacuolar protein sorting 35 (Vps35) 6
1.6 Phostensin 8
1.7運鐵蛋白循環(Transferrin recycling) 9
第二章 實驗動機與目的 10
第三章 材料與方法 12
3.1實驗材料 12
3.1.1藥品與器材 12
3.1.2儀器與套組 13
3.1.3質體 13
3.1.4引子(primer) 13
3.1.5溶液配置 14
3.2實驗方法 17
3.2.1抽取小量質體DNA(Mini plasmid DNA purification) 17
3.2.2抽取中量質體DNA(Midi plasmid DNA purification) 18
3.2.3 聚合酶連鎖反應(PCR) 19
3.2.4 PCR clean up 19
3.2.5 限制酶切割(Restriction enzyme digestion) 20
3.2.6 接合作用(Ligation) 20
3.2.7 轉型作用(Transformation) 20
3.2.8菌落聚合酶連鎖反應 (Colony PCR) 21
3.2.9 小量表達目標蛋白 22
3.2.10鎳離子瓊脂糖(Nickel chelate affinity chromatography)製備 23
3.2.11榖胱甘肽瓊脂糖(Glutathione Sepharose)製備 23
3.2.12膠體過濾層析管柱(Gel filtration chromatography)製備 23
3.2.13 低溫大量表達目標蛋白 24
3.2.14法式高壓破碎機(French press)破菌 24
3.2.15以鎳離子瓊脂糖(Nickel chelate affinity chromatography)純化目標蛋白 25
3.2.16以榖胱甘肽瓊脂糖(Glutathione Sepharose)純化目標蛋白 25
3.2.17以膠體過濾層析管柱(Gel filtration chromatography)純化目標蛋白 26
3.2.18遠西方墨點法(Far western blotting) 26
3.2.19細胞轉染(Transfection) 27
3.2.20運鐵蛋白循環試驗(Transferrin recycling assay) 28
3.2.21蛋白質定量分析(Bradford protein assay) 29
3.3.22西方點墨法(Western blotting) 30
第四章 實驗結果 31
4.1 His6-Vps35和His6-Vps35_mut的質體建構與表達 31
4.2 His6-Vps35和His6-Vps35_mut蛋白質大量純化 32
4.3 GST-EHD1和GST-EHD4蛋白質大量純化 33
4.4以遠西方點墨法探討Vps35與EHD1交互作用的影響 34
4.5以遠西方點墨法探討Vps35與EHD4交互作用的影響 34
4.6以西方點墨法分析細胞轉染後PTS-在細胞中的表達 35
4.7以流式細胞儀分析PTS-對運鐵蛋白在胞吞作用中的影響 36
第五章 討論 37
第六章 參考文獻 40
第七章 附圖 43
第八章 附錄 55
附錄A 囊泡出芽和融合的步驟圖。 55
附錄B 哺乳類細胞中的胞吞途徑。 56
附錄C EHD蛋白結構序列圖。 57
附錄D Phostensin與human Vps35 的EHD結合模序位置比較圖。 58
附錄E Vps35的EHD結合模序突變定序比對圖。 59
附錄F Retromer結構圖。 60
附錄G PTS-的EHD結合模序突變比對圖。 61
附錄H Flow cytometry analysis 62
附錄I 以遠西方點墨法探討Vps35與EHD1交互作用的影響。 63
附錄J 以遠西方點墨法探討Vps35與EHD4交互作用的影響。 64


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