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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
| dc.contributor.author | Jia-Ling Shih | en |
| dc.contributor.author | 施佳伶 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:42:01Z | - |
| dc.date.available | 2013-09-11 | |
| dc.date.copyright | 2008-09-11 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-31 | |
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King, et al. (1998). 'Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.' Nat Genet 20(1): 46-50. Neumann, G., M. T. Hughes, et al. (2000). 'Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1.' EMBO J 19(24): 6751-8. O'Neill, R. E. and P. Palese (1995). 'NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein.' Virology 206(1): 116-25. Perales, B., J. J. Sanz-Ezquerro, et al. (2000). 'The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis.' J. Virol. 74(3): 1307-1312. Perez-Gonzalez, A., A. Rodriguez, et al. (2006). 'hCLE/CGI-99, a human protein that interacts with the influenza virus polymerase, is a mRNA transcription modulator.' J Mol Biol 362(5): 887-900. Rajagopal, S. and J. Treanor (2007). 'Pandemic (avian) influenza.' Semin Respir Crit Care Med 28(2): 159-70. Regan, J. F., Y. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40163 | - |
| dc.description.abstract | 流行性感冒病毒屬於正黏液科(Orthomyxoviridae)的RNA病毒，大小約為80~120nm，具有八段負股的RNA，分別是-PB2、PB1、PA、HA、NP、NA、M、NS，其中PB2、PB1、PA、NP則是參與流感病毒複製的主要蛋白質。而流感病毒不同於其他RNA病毒的地方，在於流感病毒感染宿主細胞後會進入細胞核中進行複製，而一般RNA病毒則是在細胞質中複製。
本文的研究著重於在流行性感冒病毒在細胞內的複製機轉，以及宿主細胞因子對流感病毒的複製是否會造成影響。因為已經有許多研究指出，宿主細胞蛋白質會跟病毒蛋白質結合並影響病毒的複製過程。由於PA的功能目前尚未被完整定義，為了找出可以跟PA有交互作用的細胞蛋白質，我們初步的實驗是利用酵母菌雙雜交的方式，發現參與在流感病毒複製的PA subunit會跟HAX1、HNRPM、hCLE、SIVA、FTH1、PRDX1、MMAB等七個基因所encode的蛋白質進行交互作用。為了觀察HAX1與HNRPM是否真正參與在病毒的複製過程中，我們利用lenitivirus包裹shRNA 感染目標細胞，藉此抑制細胞中HAX1或HNRPM基因mRNA的表現。在HAX1或HNRPM基因的表現即使被抑制卻不影響細胞生長的前提下，接著以流感病毒WSN33病毒株感染基因表現被抑制的細胞株，觀察是否會影響A型流感病毒在細胞內的複製能力。 在H1299_shHAX1細胞中，可以發現HAX1基因表現被抑制之後，流感病毒vRNA的複製有稍微受到影響，但不影響病毒mRNA的合成。而在NPCTW04_HNRPM細胞中，則發現抑制HNRPM之後，病毒的vRNA與mRNA合成都降低，表示病毒的複製及轉錄能力均受到影響。因此推測，這兩個基因有可能藉著和PA之間的交互作用，來影響病毒的複製過程。但PA在人類細胞中是否真的會與HAX1或HNRPM結合，以及抑制的機轉為何都還需要再研究。 | zh_TW |
| dc.description.abstract | Influenza virus is a member of Orthomyxoviridae RNA virus which size is about 80~120nm. Influenza A virus encode eight negative-strand RNA segment which are PB2, PB1, PA, HA, NP, NA, M, NS. Different to other RNA virus, influenza A virus replicates in the nucleus. The four influenza A viral proteins, PB1, PB2, PA, and NP, comprise viral RNA replication complex, and are absolutely required for viral RNA replication and transcription. Among them, the function of PA in influenza A viral RNA replication is least known.
Several cellular proteins have been shown to interact with the subunits of influenza A RNA polymerase complex. The interaction between some cellular factors and viral replication complex has been shown to be essential for viral replication and transcription. In this study, we aimed to identify cellular proteins that can interact with the PA protein of influenza A virus by using yeast two-hybrid screening. We found that seven cellular proteins, including HAX1, HNRPM, SIVA, hCLE, FTH1, PRDX1, MMAB, might interact with PA protein. Among them, hCLE was reported previously to interact with PA. To study whether PA-interacting proteins, HAX1 and HNRPM, were involved in influenza A viral replication and transcription, we knocked down HAX1 or HNRPM expression in the cells by lentivirus expressing HAX1 or HNRPM siRNA. We found that knockdown of HAX1 and HNRPM did not affect cell growth. Influenza A vRNA, but not viral mRNA, synthesis was reduced in HAX1-knockdown cells, suggesting HAX1 is involved in influenza A viral RNA synthesis. As regard to HNRPM, we found that both vRNA and viral mRNA synthesis is reduced in HNRPM-knockdown cells, suggesting that HNRPM is involved in influenza A viral replication and transcription. How HAX1 and HNRPM affect influenza A viral replication and transcription and whether PA indeed interacts with HAX1 and HNRPM in the cells remains to investigated. The elucidation of these issues may shed light on the mechanism of influenza A replication. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:42:01Z (GMT). No. of bitstreams: 1 ntu-97-R95445113-1.pdf: 735217 bytes, checksum: df7fa921c276e21d59a01be95f8ae19f (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 目 錄 iv 圖 目 錄 vii 表 目 錄 viii 緒 論 1 一、 流行性感冒的歷史演變 1 二、 流行性感冒病毒的特徵與結構 1 三、 A型流感病毒的複製過程 2 四、 細胞因子在流感病毒複製過程中所的扮演的角色 3 五、 PA蛋白質在流感病毒複製過程中可能扮演的角色 4 研究目的 6 實驗材料 7 一、 質體 (Plasmid) 7 二、 細胞株 (Cell line) 7 三、 化學藥品與試劑 (Chemical reagents) 8 四、 套組試劑 (Kit) 10 五、 引子 (Primer) 11 六、 其他 11 實驗方法 12 一、 質體的構築 (Construction) 12 二、 勝任細胞的製作 (Competent cell preparation) 12 三、 小量質體的製備 (Mini-preparation) 13 四、 大量質體的製備 (Maxi-preparation) 14 五、 細菌轉型 (Transformation) 15 六、 酵母菌轉型 (Yeast Transformation) 16 七、 大規模酵母菌轉型及基因庫篩選 (Large scale yeast transformation and cDNA library screening) 17 八、 質體轉染 (Transfection) 18 九、 細胞核醣核酸萃取 (RNA extraction) 19 十、 反轉錄反應 (Reverase transcription) 19 十一、即時聚合酶鏈鎖反應 (Realtime-PCR) 20 十二、製作可抑制目標基因表現的去毒性慢病毒 (Preparation of pseudo-lentivirus with shRNA plasmid) 20 十三、慢病毒之感染及混合細胞株的篩選 (Lentivirus infection and pool clone selection) 20 十四、細胞生長活性之測定 (SRB Cell proliferation assay) 21 十五、A型流行性感冒病毒之製備 (Influenza virus A production) 21 十六、病毒溶斑計數實驗 (Plaque assay) 21 實驗結果 23 一、 利用酵母菌雙雜交法發現七種不同的細胞蛋白質可和PA蛋白質進行交互作用 23 二、 選擇可有效抑制HAX1及HNRPM基因表現的shRNA並製作穩定抑制基因表現的混合細胞株 23 三、 HAX1及HNRPM基因表現被抑制的混合細胞的細胞生長速率與parental細胞株及control細胞株類似 24 四、 抑制HAX1或HNRPM基因表現可能會使A型流行性感冒病毒在細胞中的複製能力下降 24 討 論 26 參考文獻 43 | |
| dc.language.iso | zh-TW | |
| dc.subject | HNRPM | zh_TW |
| dc.subject | 流感 | zh_TW |
| dc.subject | RNA病毒 | zh_TW |
| dc.subject | PA蛋白質 | zh_TW |
| dc.subject | 細胞因子 | zh_TW |
| dc.subject | RNA聚合酶 | zh_TW |
| dc.subject | HAX1 | zh_TW |
| dc.subject | influenza | en |
| dc.subject | HNRPM | en |
| dc.subject | HAX1 | en |
| dc.subject | RNA-dependent RNA polymerase | en |
| dc.subject | cellular factor | en |
| dc.subject | PA | en |
| dc.subject | RNA virus | en |
| dc.title | 鑑定並分析與A型流行性感冒病毒之PA蛋白作用的細胞因子 | zh_TW |
| dc.title | Identification and characterization of cellular factors interacting with influenza A virus PA protein | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),高全良(Chuan-Liang Kao) | |
| dc.subject.keyword | 流感,RNA病毒,PA蛋白質,細胞因子,RNA聚合酶,HAX1,HNRPM, | zh_TW |
| dc.subject.keyword | influenza,RNA virus,PA,cellular factor,RNA-dependent RNA polymerase,HAX1,HNRPM, | en |
| dc.relation.page | 47 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| 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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