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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張世宗 | |
| dc.contributor.author | Yu-En Chiu | en |
| dc.contributor.author | 邱宇恩 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:43:31Z | - |
| dc.date.available | 2023-08-21 | |
| dc.date.available | 2021-05-19T17:43:31Z | - |
| dc.date.copyright | 2018-08-21 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-17 | |
| dc.identifier.citation | 1. Taubenberger JK & Morens DM (2008) The pathology of influenza virus infections. Annu Rev Pathol 3:499-522.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7434 | - |
| dc.description.abstract | 已知A型流感病毒的PB1-F2蛋白質會調控病毒PB1聚合酶活性，亦會進入宿主細胞粒線體內外膜之間，促使膜電位下降，引發單核球細胞凋亡、調控細胞先天性免疫反應。此外PB1-F2也會抑制干擾素的生成，並增加二次細菌性肺炎感染的風險。然而，來自不同病毒株的PB1-F2在不同宿主細胞中的定位、表現量和功能都有不少差異，因此研究PB1-F2的穩定性、功能及調控機制，也許可以進一步了解不同流感病毒株致病力有所差異的原因。本研究將四株不同亞型的病毒株A/Puerto Rico/8/1934 (H1N1)、A/Udorn/307/1972 (H3N2)、A/Hong Kong/156/1997 (H5N1)、A/Taiwan/01/2013 (H7N9)的PB1-F2基因於HEK293細胞中表現時，發現其表現量有極大的差異，且皆會因加入蛋白酶體抑制劑MG132而有不同程度的上升，表示PB1-F2會受到蛋白酶體降解系統的調控。將四株不同亞型病毒株的PB1-F2進行部分胺基酸序列互換時，發現2、10、11、14和68-71會影響H1N1、H3N2和H7N9 PB1-F2在HEK293細胞中的穩定性，然而對於H5N1則沒有顯著影響。此外，細胞影像的結果顯示PB1-F2 68-71的胺基酸序列會影響PB1-F2在細胞中的分佈位置，其中ILVF有粒線體標的功能，會使PB1-F2位於粒線體。為了探究PB1-F2的穩定性是否受到泛素化的調控，將四株病毒株之PB1-F2上的所有離胺酸突變為精胺酸後，發現H1N1、H3N2、H7N9之PB1-F2皆因泛素化位點的突變而提升的在細胞中的穩定性，而H5N1 PB1-F2的穩定性則沒有因突變而有顯著提升，因此可知PB1-F2可經由泛素依賴型 (dependent) 或不依賴型 (independent) 路徑而被蛋白酶體降解。此外，分別將PA28α、PA28β或PA28γ與PB1-F2共轉染至HEK293細胞中，四株病毒株的PB1-F2的表現量皆有非常明顯的下降，顯示PA28可以促進PB1-F2的降解。 | zh_TW |
| dc.description.abstract | Influenza A virus (IAV) protein Polymerase basic 1-frame 2 (PB1-F2) regulates viral polymerase activity, induces apoptosis in host immune cells, interferes the host innate immune response and enhances the pathogenesis of secondary bacterial pneumonia. Moreover, PB1-F2 derived from different virus strains may perform different functions, expression levels and cellular localization, leading to their various strain-specific virulence in host cells. Therefore, studying the principles which determine the stability, functions and regulation mechanisms of PB1-F2 might help us know further about the pathogenesis of various influenza A virus strains. In this study, HEK293 cells expressed PB1-F2 from four IAV strains (A/Puerto Rico/8/1934 (H1N1), A/Udorn/307/1972 (H3N2), A/Hong Kong/156/1997 (H5N1), A/Taiwan/01/2013 (H7N9)) in extremely various expression levels. Moreover, the expression levels of PB1-F2 were increased upon MG132 treatment and showed different sensitivity to MG132, indicating that PB1-F2 may undergo proteasome-mediated degradation. Swapping of equivalent amino acid residues 2, 10, 11, 14, and 68-71 of PB1-F2 among these four IAV strains may alter their protein stability. Moreover, the cell images showed that amino acid residues 68-71 might modulate the localization of PB1-F2. In addition, PB1-F2 residues I68, L69, V70, and F71 are mitochondrial targeting sequence. In order to clarify whether PB1-F2 stability is ubiquitination-mediated, all of the lysine residues of PB1-F2 were mutated to arginine residues to inhibit the possible ubiquitination of PB1-F2. It is found that the expression levels of H1N1, H3N2 and H7N9 PB1-F2 were greatly increased, but H5N1 PB1-F2 did not increase significantly, indicating that the ubiquitin-dependent and ubiquitin-independent degradation pathway are all involved in regulation of PB1-F2 stability. Furthermore, the expression levels of PB1-F2 were markedly decreased while the proteasome activator PA28α/PSEM1, PA28β/PSME2, or PA28γ/PSME3 was co-transfected with PB1-F2, suggested that PA28 can promote degradation of PB1-F2. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:43:31Z (GMT). No. of bitstreams: 1 ntu-107-R05b22035-1.pdf: 4015700 bytes, checksum: e76175bc2bf02ffb3335be2abedda6b9 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 目錄 I
摘要 1 ABSTRACT 2 縮寫表 3 第一章 緒論 4 1.1 A型流感病毒 4 1.1.1 H1N1病毒 4 1.1.2 H3N2病毒 4 1.1.3 H5H1病毒 5 1.1.4 H7N9病毒 5 1.2 Polymerase basic protein 1-frame 2 (PB1-F2) 5 1.2.1 PB1-F2的發現 5 1.2.2 PB1-F2的蛋白質結構 6 1.2.3 PB1-F2參與細胞凋亡機制 6 1.2.4 PB1-F2與PB1結合調控RdRP聚合酶活性 7 1.2.5 PB1-F2調控細胞先天性免疫功能 7 1.2.6 PB1-F2提升宿主二次性細菌感染的機率 8 1.2.7 PB1-F2的穩定度 8 1.2.8 PB1-F2依賴蛋白酶體的降解機制 8 1.2.9 PB1-F2的磷酸化與泛素化 9 1.3 蛋白酶體降解機制 10 1.3.1 泛素依賴型降解路徑 10 1.3.2 泛素不依賴型降解路徑 10 1.3.3 蛋白酶體活化調節因子PA28 10 1.4研究動機 11 第二章 材料與方法 13 2.1 PB1-F2與PA28基因來源 13 2.2 大腸桿菌菌株 13 2.2.1 大腸桿菌DH5α 13 2.2.2 大腸桿菌BL21 (DE3) 13 2.2.3 大腸桿菌Rosetta (DE3) 14 2.3 人類細胞株 14 2.4 PB1-F2重組蛋白質表現質體建構 14 2.4.1 PB1-F2位點互換突變株建構 15 2.4.2 PB1-F2泛素化位點突變株建構 15 2.5 重組蛋白質表現 16 2.5.1 PB1-F2重組蛋白質表現 16 2.5.2 20S突變蛋白酶體表現 16 2.6 重組蛋白質純化 16 2.6.1 PB1-F2重組蛋白質純化 16 2.6.2 20S DM蛋白酶體純化 17 2.6.3 蛋白質脫鹽與濃縮 17 2.6.4 蛋白質定量 18 2.7 細胞培養 18 2.8 細胞轉染 18 2.9 細胞樣本製備 19 2.10 免疫染色分析 19 2.11 胞外20S蛋白酶體降解試驗 19 2.12 免疫螢光染色與雷射共軛焦掃描顯微鏡 20 第三章 結果 21 3.1 PB1-F2與20S蛋白酶體之胞外試驗 21 3.2 PB1-F2在HEK293細胞中的表現量、穩定性以及降解機制 21 3.2.1 PB1-F2在HEK293細胞中的表現量 21 3.2.2 PB1-F2在HEK293細胞中的穩定性 21 3.2.3 PB1-F2突變株在HEK293細胞中的表現量和穩定性 22 3.2.4 PB1-F2之泛素化位點突變增加其在HEK293細胞中的穩定性 23 3.2.5 PA28參與PB1-F2的降解 23 3.2.6 PB1-F2於細胞中的分佈位置 24 第四章 討論 26 4.1 PB1-F2與20S蛋白酶體之胞外試驗 26 4.2 PB1-F2重組蛋白質在細胞內的表現量和穩定性 26 4.3 PB1-F2重組蛋白質之胺基酸序列影響其在細胞內的表現量和穩定性 27 4.4 PB1-F2重組蛋白質在細胞內的降解機制 27 4.5 胺基酸序列影響PB1-F2在細胞中的分佈位置 29 4.6 A型流感病毒株之致病力與PB1-F2之穩定度 29 第五章 總結 31 參考文獻 33 圖與表 37 附錄 51 | |
| dc.language.iso | zh-TW | |
| dc.title | A型流感病毒PB1-F2蛋白質於人類細胞株之穩定性與蛋白酶體降解效率之研究 | zh_TW |
| dc.title | Investigation of the Stability and Proteasome Degradation Efficiency of Influenza A Virus PB1-F2 Proteins from Different Viral Strains in Human Cell Lines | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳慧文,張麗冠,林翰佳 | |
| dc.subject.keyword | 流感病毒,PB1-F2,蛋白?體,PA28, | zh_TW |
| dc.subject.keyword | Influenza,PB1-F2,Proteasome,PA28/PSME, | en |
| dc.relation.page | 59 | |
| dc.identifier.doi | 10.6342/NTU201803869 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2023-08-21 | - |
| 顯示於系所單位： | 生化科技學系 | |
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