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研究生:黃鈞煜
研究生(外文):Chun-Yu Huang
論文名稱:人類冠狀病毒OC43和229E核殼蛋白之結構域功能分析
論文名稱(外文):The Studies of Functional Domain of Human Coronavirus OC43 and 229E Nucleocapsid Proteins
指導教授:侯明宏
指導教授(外文):Ming-Hon Hou
學位類別:碩士
校院名稱:實踐大學
系所名稱:食品營養與保健生技研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:80
中文關鍵詞:冠狀病毒核殼蛋白
外文關鍵詞:coronavirusnucleocapsid protein
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人類冠狀病毒OC43(human coronavirus OC43, HCoV-OC43)與229E(HCoV-229E)已被證實會引起急性人類上呼吸道感染,並引發約3成的ㄧ般流行性感冒。而在冠狀病毒主要結構蛋白中,核殼蛋白(N protein)主要功能為與病毒RNA形成螺旋狀的核殼結構(nucleocapsid structure),進一步組成核醣核殼蛋白複合體(ribonucleoprotein, RNP)而形成病毒的核心,在冠狀病毒複製與轉錄的過程中扮演極重要的角色。本篇論文主要是利用不同的生化分析法,以探討人類冠狀病毒OC43與229E核殼蛋白結構域之功能,並比較二者間之差異。而在生化分析方面包含下列幾項:(1)利用電泳遷移移動分析(Electrophoretic mobility shift assay, EMSA)、螢光光譜(Fluorescence spectra)及生物分子交互作用分析系統(Biomolecular interaction analysis, BIAcore)分析此段蛋白質與核醣核酸(RNA)間之交互作用。(2)利用化學連結分析法(Chemical cross-linking assay)與凝膠過濾法(Gel filtration)觀察其自我聚合(Self-association)特性,並觀察dithiothreitol (DTT)存在對其聚合作用的影響。(3)利用圓二色光譜(Circular dichroism, CD, spectra)分析其二級結構特性。由EMSA及BIAcore的結果可知,人類冠狀病毒OC43核殼蛋白殘基174-232aa會與RNA結合,且由圓二色光譜儀分析之結果可知此段蛋白與RNA結合後,二級結構有所改變。從化學連結分析法的結果可知,在DTT存在下,HCoV-OC43核殼蛋白殘基301-448aa聚合(polymerization)情形會增強;但當HCoV-229E核殼蛋白殘基263-389aa在DTT存在下,其聚合作用則比反應前弱,這樣的結果也由凝膠過濾分析試驗得到驗證。此外,從圓二色光譜分析結果可知,HCoV-OC43核殼蛋白殘基301-448aa之二級結構以α-helix為主;而HCoV-229E核殼蛋白殘基263-389aa之二級結構構型不會因DTT的存在而有所影響。故本篇論文針對人類冠狀病毒OC43與229E核殼蛋白不同結構域之功能性已獲致初步研究結果,並將應用於此二蛋白質之未來研究,例如,功能性區域(functional domains)之晶體結構研究。
The two human coronaviruses, OC43 (HCoV-OC43) and 229E (HCoV-229E), were previously proven to cause human “common cold” and these two viruses are responsible for 30% of all the clinical cases occurring during the winter-spring time. The primary function of nucleocapsid protein (N protein), one of the structural proteins, is to recognize a stretch of RNA that serves as a packing signal leading to the formation of the ribonucleoprotein (RNP) complex during assembly. In this study, we have analyzed the functional domains of both HCoV-OC43 and 229E N proteins and the biochemical results are described as follows. First, identification of RNA binding activity by electrophoretic mobility shift assay (EMSA), fluorescence spectra and biomolecular interaction analysis (BIAcore). Second, characterization of the self-association. Third, the analyses of the secondary structures of functional domains of HCoV-OC43 and 229E N proteins by circular dichroism (CD) spectra. According to our results, in HCoV-OC43 N protein the region containing residues 174-232aa was shown to be the RNA-binding domain and the secondary structure of this region was changed upon interaction with RNA. In addition, the self-association domains of HCoV-OC43 and 229E N proteins were located at residues 301-448aa and 263-389aa, respectively. Results from CD spectra showed that α-helix being the major motif of the self-association domain of HCoV-OC43 and 229E N protein. The presence of reducing agent, dithioreitol (DTT), was shown to increase the polymerization of HCoV-OC43 N protein residues 301-448aa but the polymerization of HCoV-229E N protein residues 263-389aa was decreased in the presence of DTT. By using gel filtration, we also observed that HCoV-OC43 N protein residues 1-448aa, 301-448aa, and residues 263-389aa of HCoV-229E N protein forms 12mers, 10mers and 10 mers, respectively. The results presented here hopefully provide a promising basis for future studies such as the crystal structures of these functional domains.
目錄
目錄……………………………………………………………………………………Ι
圖目錄…………………………………………..……………………………………Ⅴ
表目錄…………………………………………..……………………………………Ⅶ
英文摘要………………………………………..……………………………………Ⅷ
中文摘要………………………………………..……………………………………Ⅹ

第一章 緒論……………………………………………………………...…..P.1
一、 冠狀病毒科(Coronaviridae)……………….………………...…..P.1
二、 冠狀病毒屬(Coronavirus)…………………………………...…..P.1
三、 冠狀病毒之結構蛋白…………………………………….……...…..P.3
四、 核殼蛋白(Nucleocapsid protein)……………………………...….. P.4
五、 實驗動機………………………………………………………….…..P.5
第二章 材料與方法……………………………………………………...…..P.7
一、 實驗材料……………………………………………….………...…..P.7
1.藥品……………………………………………………………...…..P.7
1.1.ㄧ般化學藥品…………………………………………….....…..P.7
1.2.有機溶劑………………………………………………….....…..P.8
2.載體(Vector)及勝任細胞(Competent cell)…………………..…..P.8
2.1.載體…………………………………………...……………..…..P.8
2.2.勝任細胞………………………………………...…………..…..P.8
2.3.抗生素………………………………………...……………..…..P.8
3.電泳緩衝液(Buffer)、染劑(Stain)與相關材料……………..…..P.8
3.1.瓊脂明膠(Agarose gel)…………………………………..…..P.9
3.2.蛋白質聚丙烯胺膠體(SDS-PAGE)………………..…….…..P.9
二、 實驗之儀器設備……………………………….……..…………..…..P.9
三、 實驗流程………………………………………………………..…..P.10
四、 實驗方法…………………………………………..…..………..…..P.10
1.利用PONDR® program分析HCoV-OC43與229E核殼蛋白之ordered / disordered區域……………………..…………..……P.10
2.基因選殖………………..…………………………………….……P.11
3.蛋白質粗萃液之取得………………..…………..………………...P.11
4.蛋白質純化……………….. …………………………………..…..P.12
4.1.鎳親合性管柱層析法(Ni-NTA column chromatography)..P.12
4.2.去鹽管柱(Desalting column)層析法………..…………..…..P.13
4.3.蛋白質保存………………..………………………...……..…..P.14
4.4.蛋白質濃縮………………..………………………...……..…..P.14
4.5.蛋白質定量………………..………………………...……..…..P.14
5.凝膠過濾法(Gel filtration)………………..…………...……..…..P.15
6.化學連結分析法(Chemical cross-linking assay)…...……..…..P.17
7.圓二色光譜(Circular dichroism, CD, spectroscopic analyses)分析.………..……………...…..…………...……..……...……..…..P.18
8.螢光光譜(Fluorescence spectroscopic analyses)分析....……....P.20
9.生物分子交互作用分析系統(Biomolecular interaction analysis, BIAcore)………………..………………………….....……..…..P.22
第三章 實驗結果………………..………………………...…………....…..P.26
ㄧ、 HCoV-OC43核殼蛋白………………..………….……...……..…..P.26
1.HCoV-OC43核殼蛋白(Full length)…………………..……..…..P.26
1.1.蛋白質純化………………..………………………...……..…..P.26
1.2.凝膠過濾法………………..………………………...……..…..P.26
1.3.DNA/RNA結合活性……..………………………...……..…...P.27
1.4.截取短片段蛋白……..………………………...……..………..P.27
2.HCoV-OC43核殼蛋白殘基174-232aa……..……..…...……..…..P.27
2.1.蛋白質純化……..…………………………………...……..…..P.28
2.2.RNA結合活性……..………………………... ……..……..…..P.28
2.3.圓二色光譜分析……..……………………………...……..…..P.28
3. HCoV-OC43核殼蛋白殘基233-448aa……..………...……..…..P.29
3.1.蛋白質純化……..…………………………………...……..…..P.29
3.2.化學連結分析法……..……………………………...……..…..P.29
4.HCoV-OC43核殼蛋白殘基301-448aa……..…………….…..…..P.30
4.1.蛋白質純化………………..………………………...……..…..P.30
4.2.化學連結分析法……..………………………...…………..…..P.31
4.3.凝膠過濾法……..…………..……. ……..………………...…..P.31
4.4.圓二色光譜分析………..…………………….……..……..…..P.31
二、 HCoV-229E核殼蛋白……..……………………………..……..…..P.32
1.HCoV-229E核殼蛋白殘基263-389aa……..…………..……..…..P.32
1.1.蛋白質純化……..…………..………………………….…..…..P.32
1.2.化學連結分析法……..…………..……..……………………...P.33
1.3.凝膠過濾法……..…………..……………………………...…..P.33
1.4.圓二色光譜分析…………..……………………………...……P.34
第四章 討論………..…………………………………………………...…..P.35
一、 序列比對(Alignment)………..……………………………....…..P.35
二、 PONDR® program分析………..……………………………....…..P.35
三、 HCoV-OC43核殼蛋白………..……………………………...…….P.36
1.RNA結合活性………..……………………………...………...…..P.36
2.自聚合作用(Self-association)………..…………………….…..P.37
四、 HCoV-229E核殼蛋白………..……………………………...……...P.39
1.自聚合作用(elf-association)………..……………………...…..P.39
第五章 結論與未來展望………..……………………………………...…..P.41
第六章 參考文獻………..……………………………………………...…..P.42
第七章 圖次………..…………………………………………………...…..P.45






















圖目錄

圖 1. HCoV-229E、HCoV-OC43與SARS-CoV核殼蛋白之胺基酸序列比對。...P.45
圖 2. HCoV-OC43核殼蛋白截取片段分佈位置圖。(A)HCoV-OC43核殼蛋白之PONDR® program分析圖。(B)HCoV-OC43核殼蛋白截取片段示意圖。.........P.46
圖 3. HCoV-229E核殼蛋白截取片段分佈位置圖。(A)HCoV-229E核殼蛋白之PONDR® program分析圖。(B) HCoV-229E核殼蛋白截取片段示意圖。.........P.47
圖 4. HCoV-OC43核殼蛋白純化後之電泳膠分析圖。.........................................P.48
圖 5. 以裝填SephacrylTM S-200 HR基質(分析範圍:5×103-2.5×105)的XK16/70管柱進行凝膠過濾法分析HCoV-OC43核殼蛋白(2μM)之流洗曲線圖。.......P.49
圖 6. 以螢光光譜儀分析HCoV-OC43核殼蛋白(約1μM)分別與DNA(2μM)及RNA(2μM)之結合作用。....................................................................................P.50
圖 7. HCoV-OC43核殼蛋白殘基174-232aa純化後之電泳膠分析圖。................P.51
圖 8. 利用BIAcore 3000偵測HCoV-OC43核殼蛋白殘基174-232aa(10μM)分別與RNA(20μM)及DNA(20μM)結合—解離反應之感應圖。...........................P.52
圖 9. HCoV-OC43核殼蛋白殘基174-232aa(15μM)與RNA(20.66μM)結合前後之圓二色光譜分析圖。.........................................................................................P.53
圖 10. HCoV-OC43核殼蛋白殘基233-448aa純化後之電泳膠分析圖。...........P.54
圖 11. HCoV-OC43核殼蛋白殘基233-448aa(10μM)聚合現象分析圖。......P.55
圖 12. HCoV-OC43核殼蛋白殘基301-448aa純化後之電泳膠分析圖。...........P.56
圖 13. HCoV-OC43核殼蛋白殘基301-448aa(20μM)聚合現象分析圖。......P.57
圖 14. 以裝填SephacrylTM S-100 HR基質(分析範圍:1×103-1×105)的XK16/70管柱進行凝膠過濾法分析HCoV-OC43核殼蛋白殘基301-448aa之流洗曲線圖。.............................................................................................................................P.58
圖 15. HCoV-OC43核殼蛋白殘基301-448aa(10μM)之圓二色光譜分析圖。....P.59
圖 16. HCoV-229E核殼蛋白殘基263-389aa純化後之電泳膠分析圖。...............P.60
圖 17. HCoV-229E核殼蛋白殘基263-389aa(10μM)聚合現象分析圖。........P.61
圖 18. HCoV-229E核殼蛋白殘基263-389aa(10μM)聚合現象分析圖。........P.62
圖 19. 以裝填SephacrylTM S-100 HR基質(分析範圍:1×103-1×105)的XK16/70管柱進行凝膠過濾法分析還原劑(DTT)的存在對HCoV-229E核殼蛋白殘基263-389aa聚合作用的影響。...................................................................................P.63
圖 20. HCoV-229E核殼蛋白殘基263-389aa與DTT反應前後之圓二色光譜分析圖。.............................................................................................................................P.64
圖 21. HCoV-OC43核殼蛋白功能性區域示意圖。...............................................P.65
圖 22. HCoV-229E核殼蛋白功能性區域示意圖。...............................................P.66

附圖 1. 以EMSA分析HCoV-OC43核殼蛋白殘基174-232aa與長鏈RNA之結合作用。.....................................................................................................................P.68














表目錄

表 1. HCoV-OC43與HCoV-229E核殼蛋白截取片段之物理特性。.....................P.67
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