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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77963完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉秀慧(Shiou-Hwei Yeh) | |
| dc.contributor.author | Li-Yang Chang | en |
| dc.contributor.author | 張立揚 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:38:25Z | - |
| dc.date.available | 2025-08-17 | |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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Diab, A., et al., Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication. Hepatology, 2017. 66(6): p. 1750-1765. 59. Suzuki, T., et al., Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome. Proc Natl Acad Sci U S A, 1989. 86(21): p. 8422-6. 60. Chen, J., et al., Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy. Sci Rep, 2015. 5: p. 16459. 61. Terré, S., M.A. Petit, and C. Bréchot, Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo. J Virol, 1991. 65(10): p. 5539-43. 62. Rosmorduc, O., et al., In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA. Hepatology, 1995. 22(1): p. 10-9. 63. Günther, S., et al., Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77963 | - |
| dc.description.abstract | 構成B型肝炎病毒(Hepatitis B virus；HBV)核殼(nucleocapsid)的核殼蛋白(HBV core protein；HBc)為一磷酸化蛋白質，透過其C端上Ser 155、Ser 162、Ser 170三個主要磷酸化位點調控HBV pregenomic RNA (pgRNA) encapsidation和DNA synthesis。近年研究指出HBc C端磷酸化在HBV生活史中呈現動態調控，HBc在進行pgRNA encapsidation前呈現高度磷酸化，而完成pgRNA encapsidation後則呈現低度磷酸化。然而對於HBc-S170如何透過磷酸化修飾調控pgRNA encapsidation及中間磷酸化轉變去磷酸化的分子機制仍不清楚。 本研究針對Ser 170磷酸化位點，探討其功能如何調控pgRNA encapsidation。利用專一辨認Ser 170位點去磷酸化之抗體C-S170，我們發現HBc-S170磷酸化位點在HBV 生活史中同樣呈現動態調控，在pgRNA encapsidation前後分別出現高度和低度磷酸化。透過回補的方式，顯示Ser170位點透過polymerase調控pgRNA encapsidation。除此之外，亦發現HBc和HBc-Cys蛋白上之Ser 170磷酸化位點均扮演調控pgRNA encapsidation進行之重要角色。本論文研究結果因此指出HBc和HBc-Cys蛋白上之Ser 170 為點之磷酸化有可能會藉由改變其蛋白質C端胺基酸序列之結構而影響其與polymerase蛋白之結合而增加pgRNA encapsidation之假說，值得未來進一步探討。 | zh_TW |
| dc.description.abstract | HBV core protein (HBc), the base subunit of Hepatitis B virus (HBV) nucleocapsid, is a phosphorylated protein regulating HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis through three major phosphorylation sites at Ser155, Ser162, Ser170 on its C-terminal domain (CTD). Recent studies have demonstrated that HBc C-terminal undergoes dynamic phosphorylation during HBV life cycle. HBc is hyperphosphorylated before pgRNA encapsidation and hypophosphorylated after pgRNA encapsidation. Nevertheless, the molecular mechanisms for the phosphorylation of HBc-Ser170 in regulating pgRNA encapsidation and the specific events occurring from phosphorylation to dephosphorylation are still unclear. This study focused on HBc-Ser170 (S170) phosphorylation site for investigating its function in regulating the RNA encapsidation process, in the replicon transfected cells. Using the dephosphorylated specific C-S170 Ab, we found a dynamic phosphorylation pattern of HBc-S170 in different replication stages, highly phosphorylated before RNA encapsidation but de-phosphorylated afterwards. By complementation assay, we demonstrated that HBV pgRNA encapsidation could be mediated through the recruited polymerase. Moreover, we found the phosphorylation of S170 is essential for both HBc and HBc-Cys proteins to proceed the RNA encapsidation process. The possibility that phosphorylation of S170 in HBc and HBc-Cys protein could be critical for recruitment of polymerase via inducing a conformational change of their C-terminal domain structure is worthy to be tested in future studies. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:38:25Z (GMT). No. of bitstreams: 1 U0001-1608202001054900.pdf: 2441770 bytes, checksum: 2c4ccea7a289e0872cb4ada143766cc5 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 碩士論文口試委員審定書 i 誌謝 ii 摘要 iii Abstract iv 目錄 - 1 - 圖表目錄 - 3 - 第一章 序論 - 4 - 1.1 Hepatitis B virus (HBV)簡介 - 4 - 1.2 HBV基因體結構 - 4 - 1.3 HBV生活史 - 5 - 1.4 HBV核殼蛋白之結構與功能 - 6 - 1.5 核殼蛋白的磷酸化位點在HBV複製中扮演角色 - 7 - 1.6 HBc 自dimer 進行組裝經 intermediate 至 virion 之過程 - 8 - 1.7 HBc 與HBV RNA polymerase作用對pgRNA encapsidation之影響 - 9 - 1.8 調控HBc磷酸化之候選kinase及phosphatase - 10 - 1.9 HBV SP1剪接RNA (splicing RNA) 之簡介 - 11 - 第二章 研究假說與策略 - 13 - 第三章 實驗材料與方法 - 14 - 3.1 質體(Plasmids) - 14 - 3.2 點突變(Site-direct mutagenesis) - 16 - 3.3 細胞培養(Cell culture) - 17 - 3.4 細胞轉染(Transfection) - 17 - 3.5 蛋白質萃取(Protein extraction) - 18 - 3.6 去磷酸化反應(Alkaline Phosphatase reaction) - 18 - 3.7.1 免疫共沉澱(Co-immunoprecipitation-Anti-Flag M2 magnetic beads) - 18 - 3.7.2免疫沉澱(Immunoprecipitation-G beads) - 19 - 3.8 西方墨點法(Western blot-SDS PAGE) - 20 - 3.9 HBV nucleocapsid偵測(Native agarose gel electrophoresis) - 20 - 3.10 HBV nucleocapsid內DNA偵測(Particle gel assay) - 20 - 3.11 萃取HBV nucleocapsid內RNA (RNA extraction) - 21 - 3.12 北方墨點法(Northern blot) - 22 - 3.13蔗糖密度梯度離心(Sucrose gradient centrifugation) - 22 - 3.14抗體(Antibodies) - 23 - 第四章 實驗結果 - 24 - 4.1 HBV在各個replication stage中HBc-S170位點之磷酸化程度 - 24 - 4.2 HBV 透過polymerase 參與RNA encapsidation 及HBc-S170位點去磷酸化 - 25 - 4.3 HBc-S170磷酸化位點透過polymerase蛋白調控去磷酸化之RNA encapsidation - 26 - 4.4 HBc-Cys與HBc之S170磷酸化位點對於polymerase蛋白調控DNA synthesis功能同等重要 - 27 - 4.5 HBc-S170透過CTD協助pol調控去磷酸化之RNA encapsidation - 28 - 4.6 HBc-S170可能透過改變CTD構型協助RNA encapsidation之進行 - 28 - 第五章 討論 - 31 - 參考文獻 - 34 - 圖表 - 41 - | |
| dc.language.iso | zh-TW | |
| dc.subject | 聚合酶 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | 核殼蛋白 | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | 包裹核酸 | zh_TW |
| dc.subject | pgRNA encapsidation | en |
| dc.subject | hepatitis B virus | en |
| dc.subject | Polymerase | en |
| dc.subject | core protein | en |
| dc.title | 探討B型肝炎病毒核殼蛋白 Serine 170 位點磷酸化調控病毒複製之影響 | zh_TW |
| dc.title | The phosphorylation of HBV core protein at Serine-170 in regulating viral RNA encapsidation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳培哲(Pei-Jer Chen),鄧述諄(Shu-Chun Teng),王聖涵(Sheng-Han Wang) | |
| dc.subject.keyword | B型肝炎病毒,核殼蛋白,磷酸化,包裹核酸,聚合酶, | zh_TW |
| dc.subject.keyword | hepatitis B virus,core protein,pgRNA encapsidation,Polymerase, | en |
| dc.relation.page | 47 | |
| dc.identifier.doi | 10.6342/NTU202003554 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-17 | - |
| 顯示於系所單位： | 微生物學科所 | |
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