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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 余明俊(Ming-Jiun Yu) | |
dc.contributor.author | Wei-Ting Kao | en |
dc.contributor.author | 高維霆 | zh_TW |
dc.date.accessioned | 2021-06-08T01:07:22Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-18 | |
dc.identifier.citation | 1 http://www.who.int/hiv/pub/hepatitis/hepatitis-c-guidelines/en/
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18480 | - |
dc.description.abstract | C型肝炎病毒(Hepatitis C virus, HCV)為單股正向RNA病毒,其基因體長度約為9.6 kb並可轉錄成一條多蛋白(polyprotein),polyprotein經由宿主以及病毒蛋白酶的切割後會產生十種不同功能的病毒蛋白,主要分成結構性蛋白(structural protein)以及非結構性蛋白(nonstructural protein)。其中非結構性蛋白5A(nonstructural protein 5A, NS5A)如何參與病毒複製的機制至今尚未明瞭,有報告指出NS5A會受磷酸化調控進而影響病毒的複製,但NS5A的關鍵磷酸化位點尚未清楚。
先前實驗室利用質譜方式鑑定出NS5A上六個不同的磷酸化位點,分別為S2198、S2201、S2205、S2208、S2211和S2214位點,其中S2198、S2211和S2214會發生磷酸化的可信度高於百分之99。為了進一步探討這三個位點,我們把帶有報導基因的HCV載體上的三個serine位點突變成alanine抑制磷酸化的發生,並表現於人類肝腫瘤細胞株中(Hul 7.5.1)。結果顯示S2198位點突變的HCV載體,其報導基因活性和野生型(wild-type)表現量一樣,顯示S2198位點的磷酸化不影響HCV的複製。而S2214位點突變株的報導活性雖隨時間增加而上升,但在轉染(transfection)72小時之後活性卻不及野生型的表現,推測S2214位點的磷酸化對病毒的複製有些微的影響。然而S2211位點的突變株,其報導基因活性會隨著時間增加而大幅度下降,轉染72小時之後幾乎測量不到報導基因活性,進而推測S2211位點發生磷酸化HCV才能夠的複製,S2211位點磷酸化對HCV的複製是非常重要的。無論在雙重位點突變株(double mutation)或三重位點突變株(triple mutation)當中,只要S2211發生磷酸化,就測量不到報導基因活性,由此可以推論出,在HCV的生活史當中,S2211位點磷酸化支配著S2198位點和S2214位點的磷酸化。 | zh_TW |
dc.description.abstract | HCV non-structural protein 5A (NS5A) is a phospho-protein critical for HCV life cycle. NS5A has hypo- and hyper-phosphorylation states; the latter has been implicated in viral replication and assembly. Despite of several attempts, precise phosphorylation sites responsible for NS5A phosphorylation states and their functions remains largely unknown. We identified six NS5A phosphorylation sites in HCV-infected hepatocarcinoma cells (Huh 7.5.1) using protein tandem mass spectrometry. Using an HCV full genome reporter construct, we made single, double, and triple alanine mutations of S2198, S2211, and S2214 sites that were identified with high confidence. The HCV RNAs were made and transfected into Huh7.5.1 cells followed by measurement of luciferase activity to assess virus activity. Phosphorylation-ablated alanine mutant at S2198 showed similar virus activity profile as the wild type, suggesting no apparent role of S2198 phosphorylation in viral life cycle. The reporter activity of the S2214A mutant failed to increase to the same levels as that of the wild type. In a sharp contrast, the virus activity of the S2211 mutant was completely abolished, indicating a major role of S2211 phosphorylation in HCV life cycle. Indeed, as long as the S2211 site was mutated in double or triple mutations, the virus activity was completely blunted, suggesting that S2211 phosphorylation dominates over S2198 and S2214 phosphorylation in genotype 2a HCV replication. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:07:22Z (GMT). No. of bitstreams: 1 ntu-103-R01442019-1.pdf: 1830389 bytes, checksum: d15fa851849d6fce8935381dfe9a3a00 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | Contents 1
誌謝 2 摘要 3 Abstract 4 Introduction 5 Materials & Methods 10 Results 19 Mutant J6/JFH1 HCV construct preparation 19 NS5A S2211 phosphorylation dominates over S2198 and S2214 phosphorylation in genotype 2a HCV replication...20 Figures 23 Figures Legends 29 Discussion 33 References 38 | |
dc.language.iso | en | |
dc.title | 鑑定C型肝炎病毒生活史中非結構性蛋白5A的優勢磷酸化位點 | zh_TW |
dc.title | Identification of a Predominant Phosphorylation Site in Non-Structural Protein 5A Involved in Hepatitis C Virus Life Cycle | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 施嘉和(Chia-Ho Shih),楊宏志(Hung-Chih Yang) | |
dc.subject.keyword | C型肝炎病毒,多蛋白,非結構性蛋白5A,野生型,轉染,雙重位點突變株,三重位點突變株, | zh_TW |
dc.subject.keyword | Hepatitis C virus,polyprotein,nonstructural protein 5A,wild-type,transfection,double mutation,triple mutation, | en |
dc.relation.page | 41 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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