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研究生:邱雅莉
研究生(外文):Ya-Li Chiu
論文名稱:人類冠狀病毒229E核殼蛋白C端於RNA結合活性及聚合能力之特性分析
論文名稱(外文):Characterization of the C-terminal domain of human coronavirus 229E nucleocapsid protein in RNA-binding and oligomerization
指導教授:侯明宏
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:68
中文關鍵詞:冠狀病毒核殼蛋白聚合作用
外文關鍵詞:coronavirusnucleocapsid proteinoligomerization
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冠狀病毒 (coronavirus) 常會引起人類傳染性呼吸及腸胃型感冒。人類冠狀病毒229E (HCoV-229E) 為單股的正股RNA病毒,其遺傳物質RNA先由核殼蛋白 (nucleocpasid protein, N protein) 包裹成長鏈螺旋狀的核糖核酸蛋白複合體 (ribonucleoprotein complex) ,再包裝入蛋白質套膜,進而組裝成完整的病毒顆粒。核殼蛋白是冠狀病毒中數量最多的結構蛋白,且在不同冠狀病毒中皆保留兩段結構功能區域 (structure domain) ,分別是N端 (N-terminal domain) 的RNA結合區域及C端 (C-terminal domain) 的自我聚合區域。HCoV-229E核殼蛋白具有389個胺基酸,本篇主要探討HCoV-229E核殼蛋白C端的特性及其末端39個胺基酸扮演的角色。首先,由disordered區域預測軟體PONDR®分析HCoV-229E核殼蛋白,依此構築C端ordered區域蛋白片段胺基酸245-350 (a.a.245-350) 及包含末端disordered區域的蛋白片段胺基酸245-389 (a.a.245-389) 。接著,以表面薄膜技術 (surface plasmon resonance,SPR) 證實本實驗所構築之HCoV-229E核殼蛋白C端不具有RNA結合活性。再以化學交聯實驗 (chemical cross-linking) 、分子篩管柱層析法 (size exclusion chromatography,SEC) 、分析型超高速離心機 (analytical ultracentrifuge, AUC) 分析HCoV-229E核殼蛋白C端兩蛋白片段 (a.a.245-350及a.a.245-389) 聚合能力的差異,發現蛋白a.a.245-350主要會聚合成雙聚體 (dimer) ,而增加了末端39個胺基酸的蛋白a.a.245-389則容易聚合成四聚體 (tetramer) 或多聚體 (multimer) 。最後,利用圓二色光譜儀 (circular dichroism, CD) 分析兩C端胜肽片段結構上的差異,並利用軟體CDsstr分析兩C端蛋白片段各二級結構的含量。由以上結果可推測HCoV-229E核殼蛋白可能是以雙聚體為基礎單位 (building block) ,蛋白a.a.245-350即有形成基礎單位的能力,再藉由C端末端disordered區域的輔助而聚合成多聚體,此特性對於冠狀病毒利用核殼蛋白將遺傳物質RNA包裹成結構較緊密的核醣核酸蛋白複合體極為重要。
Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans. Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form a long helical nucleocapsid during virion assembly. The N protein of HCoV-229E was found to self-associate through the action of its carboxyl terminus. In this study, we constructed two truncated fragments of HCoV-229E N protein, namely amino acids 245-350 (a.a. 245-350) and amino acids 245-389 (a.a. 245-389). The binding of the C-terminal domain of HCoV-229E N protein (a.a. 245-389) to RNA was analyzed by using surface plasmon resonance (SPR). The SPR results indicated that the C-terminal domain of HCoV-229E N protein lacks RNA-binding activity. To further understand the oligomerization of the C-terminal domain, chemical cross-linking assay, size exclusion chromatography, and analytical ultracentrifuge were applied. The cross-linking assay experiments by increasing the concentrations of glutaraldehyde showed that both truncated fragments, i.e., a.a. 245-350 and a.a. 245-389; were able to form dimers and tetramers, but a.a. 245-389 was the only fragment that formed octamers and high-order oligomers. In addition, the results revealed by the gel-filtration chromatography and analytical ultracentrifuge assay indicated that the removal of 39 amino acids (residues 351-389) at the C-terminus decreased the oligomerization ability of the C-terminal domain of HCoV-229E N protein (a.a. 245-389), in agreement with previous chemical cross-linking results. We proposed here that the 39 amino acids at the C-terminus are indispensable in terms of the oligomerization of HCoV-229E N protein. This study may benefit the understanding of the formation mechanism of long nucleocapsid of type I HCoV-229E.
摘要 i
Abstract iii
目次 v
表次 viii
圖次 ix
第一章、 前言 1
一、 冠狀病毒概述 1
二、 冠狀病毒感染模式 1
三、 冠狀病毒的基因體 3
四、 人類冠狀病毒及分類 4
五、 HCoV-229E與HCoV-NL63的關係 5
六、 HCoV-229E與疾病 6
七、 核殼蛋白 (N蛋白) 6
八、 研究動機 7
第二章、 材料與方法 9
一、 實驗材料 9
(一) 化學藥品及有機溶劑 9
(二) 載體 10
(三) 勝任細胞 10
(四) 實驗中使用的Kit及感應晶片 10
(五) 各種溶液與緩衝溶液 11
(六) 儀器設備 12
二、 實驗方法 12
(一) 序列分析比對 12
(二) 序列二級結構及disordered區域預測 13
(三) 載體的構築 13
(四) 菌體培養、誘導蛋白大量表現 13
(五) 蛋白純化 14
(六) 表面薄膜共振 (surface plasmon resonance, SPR) 16
(七) 化學交聯 (chemical cross-linking) 實驗 18
(八) 分子篩管柱層析法 (size exclusion chromatography, SEC) 19
(九) 分析型超高速離心機 (Analytical ultracentrifuge, AUC) 20
(一) 圓二色光譜 (circular dichroism, CD) 分析 20
三、 實驗架構 23
第三章、 結果 24
一、 核殼蛋白C端序列比對 24
二、 HCoV-229E核殼蛋白C端二級結構預測 24
三、 HCoV-229E核殼蛋白disordered區域預測 25
(一) PONDR® 25
(二) PrDOS 25
四、 HCoV-229E核殼蛋白C端片段蛋白構築 25
五、 HCoV-229E核殼蛋白C端片段蛋白純化 25
六、 HCoV-229E核殼蛋白純化分析 26
七、 RNA結合活性測試 26
八、 化學交聯實驗 27
(一) 蛋白濃度變化 27
(二) Glutaraldehyde濃度變化 27
九、 分子篩管柱層析 28
(一) 蛋白a.a.245-350 28
(二) 蛋白a.a.245-389 29
十、 分析型超高速離心機 29
(一) 蛋白a.a.245-350 29
(二) 蛋白a.a.245-389 30
十一、 圓二色光譜分析 30
第四章、 討論 31
一、 核殼蛋白C端序列比對 31
二、 核殼蛋白C端二級結構預測 31
三、 核殼蛋白disordered區域預測 32
四、 核殼蛋白C端片段蛋白構築及純化 33
五、 RNA結合活性分析 33
六、 聚合能力分析 34
七、 蛋白二級結構差異分析 38
第五章、 結論 40
第六章、 表次 41
第七章、 圖次 43
第八章、 附表 58
第九章、 附圖 60
第十章、 參考文獻 63
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