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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
| dc.contributor.author | Zih-Hsuan Huang | en |
| dc.contributor.author | 黃子軒 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:19:53Z | - |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-08 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18694 | - |
| dc.description.abstract | 流感病毒的PA、PB1及PB2蛋白質構成了病毒特有的RNA-dependent RNA polymerase (RdRp)，參與病毒複製轉錄的過程，在病毒生活史扮演重要的角色。本實驗室為了更了解病毒複製轉錄的機制，挑選PA進行更進一步的研究。首先，以PA為餌，利用酵母菌雙雜合系統(yeast two-hybrid system)篩選Hela細胞的cDNA資料庫，找出了幾個可能與PA有交互作用的細胞蛋白質，並對其中的hnRNP M進行更深入探討。
在之前的研究中，我們利用帶有tag的PA與hnRNP M蛋白質進行免疫共沉澱法(Co-IP)以及GST pull-down assay，確認兩者之間具有交互作用；在RNase A的作用下，也證實兩者之間並非透過RNA而結合。接著我們想了解此交互作用對病毒或宿主有甚麼生理意義，使用了溶斑分析法((plaque assay))和螢光酶報導系統(Luciferase reporter assay)之後，發現hnRNP M具有抑制流感病毒複製的效果。 為了進一步了解hnRNP M抑制流感病毒複製的機制，我們純化了帶有tag的PA與hnRNP M蛋白質，利用in vitro的方式，發現hnRNP M可以影響PA內切酶的活性。此外，我們也發現到當細胞受到流感病毒感染下，細胞體內的hnRNP M會大量表現。我們也進一步的發現到hnRNP M可以被干擾素α、β誘導而產生。另一方面，我們也發現細胞樣本在受到病毒感染的情況下，會有干擾素α、β的產生。 以上結果顯示，hnRNP M會藉由與PA的交互作用影響PA內切酶的活性，負向調控病毒複製轉錄的功能，而hnRNP M也會受到第一型干擾素的誘導，因此我們推論hnRNP M可能是細胞中抵抗病毒的重要成員之一。 | zh_TW |
| dc.description.abstract | Influenza A virus contains an RNA-dependent RNA polymerase(RdRp) which consists of viral proteins PA, PB1 and PB2. It plays an important role in influenza viral transcription and replication. PA had been shown to be involved in vRNA synthesis and to activate viral mRNA synthesis by regulating the ‘cap-snatching’ process. In previous studies, our lab used yeast two-hybrid system to identify cellular factors that may interact with PA protein. We found that hnRNP M, an mRNA splicing factor, is one of the candidates of PA-interacting cellular proteins.
To further confirm the interaction between PA and hnRNP M, co-immunoprecipitation (Co-IP) and GST pull-down assays were performed. The results demonstrated that PA did specifically interact with hnRNP M. Moreover, we performed plaque assay and Luciferase reporter assay to investigate the influence of hnRNP M on influenza virus replication. Both data showed that hnRNP M reduced the efficiency of virus replication. In order to figure out the mechanism of how hnRNP M reduced the efficiency of virus replication, we purified hnRNP M protein and PA protein. With in vitro assay, we found out that hnRNP M can reduce the endonuclease activity of PA. Furthermore, through western blot, we showed that the expression level of hnRNP M was induced by influenza virus infection. We also found out that hnRNP M could be induced by interferon α、β. And we also showed that interferon α、β was induced by influenza virus infection. Together, these data suggest that hnRNP M may be one of the cellular anti-viral proteins that are induced upon influenza A virus infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:19:53Z (GMT). No. of bitstreams: 1 ntu-103-R01445121-1.pdf: 2190033 bytes, checksum: 64b54833e06e5e4528326bf93bb251d1 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………....................... i
Abstract………………………………………………………………………............. ii 目錄.............................................................................................................................. iii 圖目錄...........................................................................................................................vi 緒論……………………………………………………………………………........... 1 材料與方法……………………………………………………………………........... 9 ◆ 實驗材料………………………………………………………………………..... 9 一、化學藥品及試劑………………………………………………………………... 9 二、套組試劑………………………………………………………………………... 12 三、抗體……………………………………………………………………………... 13 四、酵素……………………………………………………………………………... 13 五、其它……………………………………………………………………………... 14 六、細胞株 (Cell line)………………………………………………………………. 14 七、質體 (Plasmid)…………………………………………………………………. 15 ◆ 實驗方法………………………………………………………………………... 19 一、質體建構 (Construction)……………………………………………………….. 19 二、細菌轉形 (Transformation)…………………………………………………… 19 三、勝任細胞的製備 (Preparation of competent cells)…………………………… 19 四、小量質體製備 (Mini-preparation)………………………………………….….. 21 五、大量質體製備 (Large-scale plasmid isolation) ……………………………….. 22 六、質體轉染 (Transfection)……………………………………………………….. 24 七、慢病毒製備 (Preparation of Lentivirus)……………………………………….. 25 八、慢病毒定量 (quantification of Lentivirus) ………………………………......... 26 九、慢病毒感染 (Lentivirus infection)……………………………………………... 28 十、細胞核糖核酸萃取 (RNA extraction)…………………………………………. 28 十一、反轉錄反應 (Reverse transcription)………………………………………… 29 十二、即時聚合酶鏈鎖反應 (Real-time PCR) …………………………………..... 29 十三、半定量聚合酶鏈鎖反應 (semi-quantitative PCR)………………………..… 30 十四、螢光酶分析 (Luciferase assay)…………………………………………..….. 31 十五、細胞全蛋白質之收取……………………………………………………..…. 31 十六、蛋白質定量………………………………………………………………..…. 32 十七、西方墨點法 (Western blot)………………………………………………….. 32 十八、流感病毒感染及增殖 (Influenza virus infection and amplification)……..… 33 十九、流感病毒之溶斑分析法 (Plaque assay of Influenza virus)………………… 33 二十、Glutathione S-transferase (GST) pull-down 分析………………………….... 35 二十一、免疫共沈澱法 (Co-Immunoprecipitation)……………………………….. 37 二十二、免疫螢光分析 (Immunofluorescence assay, IFA)……………………….. 37 二十三、GST-tag 蛋白質純化................................................................................... 38 二十四、His-tag 蛋白質純化..................................................................................... 38 二十五、體外轉錄(in vitro transcription)................................................................... 39 二十六、體外內切酶活性測試(in vitro endonuclease activity assay)....................... 39 研究目的…………………………………………………………………………..... 40 實驗結果……………………………………………………………………………. 41 一、利用體外試驗(in vitro assay)確認細胞蛋白hnRNP M能抑制病毒蛋白PA的內切酶活性……………………………………………………...........................41 二、A型流感病毒能在H1299細胞中誘導hnRNP M產生.......................................41 三、A型流感病毒無法在H1299-shIFNAR1細胞中誘導hnRNP M產生................ 42 四、hnRNP M能在H1299細胞中被干擾素(interferon,IFN) α、β誘導產生............... 42 五、A型流感病毒能於H1299細胞中促使干擾素(interferon,IFN) α、β產生......... 42 討論…………………………………………………….................………………… 44 圖表............................................................................................................................. 47 附圖…………………………………………………………………………………. 59 附表…………………………………………………………………………………. 62 參考文獻……………………………………………………………………………. 63 | |
| dc.language.iso | zh-TW | |
| dc.title | 細胞蛋白hnRNP M在A型流感病毒感染下所辦演的角色 | zh_TW |
| dc.title | The role of cellular protein hnRNP M in Influenza A virus infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),楊宏志(Hung-Chih Yang) | |
| dc.subject.keyword | 流行性感冒病毒,PA蛋白質,hnRNP M,流感病毒轉錄與複製, | zh_TW |
| dc.subject.keyword | Influenza virus,PA,hnRNP M,influenza viral transcription and replication, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2014-08-08 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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