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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 | 葉秀慧 | |
dc.contributor.author | Yi-Ting Chiu | en |
dc.contributor.author | 邱宜亭 | zh_TW |
dc.date.accessioned | 2021-05-19T17:50:46Z | - |
dc.date.available | 2022-09-08 | |
dc.date.available | 2021-05-19T17:50:46Z | - |
dc.date.copyright | 2017-09-08 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7705 | - |
dc.description.abstract | B型肝炎病毒(Hepatitis B virus;HBV)之pregenomic RNA (pgRNA)可直接轉譯出核殼蛋白(HBV core protein;HBc),或經剪接(splicing)產生SP1 spRNA再轉譯出相較於wild-type HBc缺少C端最後一個胺基酸Cys183之truncated HBc (HBc’)。由於wild-type HBc與SP1 spRNA所產生的HBc’蛋白質在負責病毒nucleocapsid組裝的N-terminal domain胺基酸序列完全相同,因此推論在HBV進行複製過程中,HBc’可能會與wild-type HBc形成dimer,進一步組裝成capsid,而同時存在於HBV capsid中。然而HBV capsid中HBc及HBc’蛋白在病毒複製過程中所扮演的功能性角色有無差異仍有待進一步釐清。本研究利用點突變建構只表現HBc及HBc’的HBV replicon質體,並分析其所組成的capsid對於HBV replication cycle中各個步驟,包括RNA encapsidation、DNA複製,至後續進行包膜(envelopment)並釋出到細胞外之可能功能是否有差異。比較WT及HBc-與HBc’- HBV replicon於細胞中複製情形時,為確認細胞中capsid含量,進一步以sucrose gradient分離HBc (HBc’)之dimer form與capsid form,結果發現HBc-與HBc’- HBV replicon 之capsid相較於WT replicon有減少現象。但藉由分析細胞內capsid-RNA及capsid-DNA的表現量,發現HBc及HBc’對於RNA encapsidation,及進行後續反轉錄(reverse transcript)以產生capsid-DNA之病毒複製步驟上無顯著功能之差異。進一步探討HBc與HBc’是否會影響已經完成複製的capsid進行後續envelopment,並且釋放至細胞外之過程,結果發現HBc-HBV 與HBc’-HBV replicon釋放至細胞外的病毒顆粒有所差異,指出wild-type HBc可能參與capsid進行envelopment並釋放至細胞外的過程。本研究結果首度指出HBV capsid 中HBc及HBc’ 蛋白在病毒複製過程中可能扮演不同的角色,未來將針對HBc-與HBc’- HBV replicon 所產生之capsid減少的機制及HBc蛋白決定capsid成功envelopment之假說進行進一步探討。 | zh_TW |
dc.description.abstract | The hepatitis B virus (HBV) core protein (HBc), which consists of 183 a.a., is encoded by the viral pregenomic RNA (pgRNA). The pgRNA can be spliced to generate several splicing RNAs (spRNAs). Among them, the SP1 spRNA encodes an HBc homolog, namely HBc’, which is lack of only one cysteine residue at the very C terminal end of HBc. The HBc protein contains two functional domains, the N terminal 1-149 a.a. responsible for nucleocapsid assembly and the C terminal 150-183 a.a. attributing to the nucleic acid binding. Therefore, both HBc and HBc’ proteins contain the identical N terminal assembly domain, which could form dimer and be assembled into the capsids. It will be interesting to address if any functional difference between HBc and HBc’ proteins in capsid in the viral replication cycle. This study took the approach to generate the HBV replicon constructs, which express either HBc or HBc’ protein only, to examine the functional effect on each viral replication step, including the RNA encapsidation, reverse transcription of RNA to viral DNA, and also the envelopment/release of virion. We have also conducted the sucrose gradient analysis to examine the amount of dimer and capsids for each construct. Compared with the wild type replicon, though the capsid amount in HBc- and HBc’-replicon transfected cells is decreased, both the RNA encapsidation and the DNA synthesis is not affected by depletion of either HBc or HBc’. However, we found that the envelopment of the capsids is different between HBc-replicon and HBc’-replicon, indicating a critical role of HBc in the viral envelopment process. The preliminary results of this study indicated that the HBc and HBc’ protein in the capsid might play distinct function in viral replication cycle. It forms a basis for future investigation of the functional role and mechanism for HBc and HBc’ in determination of amount and envelopment of viral capsids. | en |
dc.description.provenance | Made available in DSpace on 2021-05-19T17:50:46Z (GMT). No. of bitstreams: 1 ntu-106-R04445106-1.pdf: 1938578 bytes, checksum: efd3de7c40382379f4fc4f5b57ca3d3e (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii 圖表目錄 vi 第一章 序論 1 1.1 Hepatitis B virus (HBV)簡介 1 1.2 HBV的生活史 1 1.3 HBV核殼蛋白之結構與功能 2 1.4 HBV RNA剪接(Splicing) 4 1.5 SP1 spRNA所轉譯之truncated HBc介紹 4 第二章 研究假說與策略 6 第三章 實驗材料與方法 7 2.1質體 7 2.2點突變 (Site-directed mutagenesis) 8 2.3細胞培養 (Cell culture) 9 2.4細胞轉染 (Transfection) 10 2.5蛋白質抽取 10 2.6細胞外病毒顆粒之純化 11 2.7 Sucrose gradient 11 2.8西方墨點法 (Western blot) 11 2.9 HBV capsid之偵測 12 2.10細胞RNA萃取 12 2.11純化 HBV nucleocapsid 內之DNA 13 2.12純化 HBV nucleocapsid 內之RNA 13 2.13北方墨點法 (Northern blot) 14 2.14南方墨點法 (Southern blot) 15 第四章 實驗結果 16 4.1建構只表現HBc及HBc'的HBV replicon質體 16 4.2分析不同抗體對HBc與HBc' protein及capsid之辨認性 16 4.3比較WT及mutant HBV replicon於細胞中複製情形 17 4.4利用Sucrose gradient比較WT及mutant HBV replicon於細胞中所生成capsid量的多寡 18 4.5比較細胞轉染WT及mutant HBV replicon後釋放至細胞外之不同病毒顆粒之特性 19 4.6破壞病毒splicing acceptor site 487/488探究HBc'之功能 21 4.7藉由Hydrodynamic injection mouse model in vivo驗證HBc'於病毒複製之功能 21 4.8 HBV capsid中wild-type HBc及HBc'功能之假說 22 4.9於HBc' replicon共轉染HBc表現質體探討HBc對capsid envelopment之重要性 23 4.10藉由抑制lysosome、autophagy降解路徑檢驗HBc'於穩定HBc-capsid之功能 24 第五章 討論 25 參考文獻 27 圖表 33 | |
dc.language.iso | zh-TW | |
dc.title | 探討B型肝炎病毒剪接RNA衍生之蛋白質對病毒複製之影響 | zh_TW |
dc.title | Investigate the Function of HBV Spliced RNA Derived Protein Product in Viral Replication | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳培哲,吳慧琳 | |
dc.subject.keyword | B型肝炎病毒,核殼蛋白,RNA 剪接,病毒複製, | zh_TW |
dc.subject.keyword | hepatitis B virus,nucleocapsid,RNA splicing,viral replication, | en |
dc.relation.page | 47 | |
dc.identifier.doi | 10.6342/NTU201703276 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2017-08-15 | |
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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