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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張明富(Ming-Fu Chang) | |
dc.contributor.author | Che-Lun Fan | en |
dc.contributor.author | 范哲綸 | zh_TW |
dc.date.accessioned | 2021-06-16T05:27:09Z | - |
dc.date.available | 2019-10-09 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-14 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56407 | - |
dc.description.abstract | B型肝炎病毒為一種含有核衣殼的小型DNA病毒,帶有3.2-kb的病毒基因體,且外圍被外套所包覆。B型肝炎病毒已在世界各地感染約20億人,其中有3億5千萬人屬於慢性感染。慢性B型肝炎病毒感染會引發嚴重的肝臟疾病,如肝硬化與肝癌。而D型肝炎病毒為B型肝炎病毒之衛星病毒,需要B型肝炎表面抗原才能組裝成D型肝炎病毒顆粒以進行感染。D型肝炎病毒感染約1千5百萬人。如果慢性B型肝炎病毒感染的患者同時感染D型肝炎病毒,會大大提高嚴重併發症之風險,並引發更快速的癌症發展。最近,有研究指出sodium taurocholate cotransporting polypeptide (NTCP)為B型與D型肝炎病毒之受體。在本研究中,表現人類NTCP以建立肝臟細胞專一性的體外B型肝炎病毒感染系統,並以體外方式製備B型與D型肝炎病毒顆粒。B型肝炎病毒顆粒可藉由HepG2.2.15細胞株穩定且持續生產。而利用猿猴病毒40早期啟動子調控表現雙套D型肝炎病毒cDNA的pSVD2質體轉染HepG2.2.15細胞株之系統則可以得到D型肝炎病毒顆粒。本研究同時發現D型肝炎病毒存在時會提高HepG2.2.15細胞株系統中B型肝炎病毒顆粒的生產量。此外,當HepG2細胞株轉染表現肝臟細胞專一性外源人類NTCP蛋白質時,可被B型肝炎病毒感染。本研究所建立之肝臟細胞專一性體外B型肝炎病毒感染系統或許可提供一簡便的平台以研究病毒感染、病毒間交互作用、致病機轉以及藥物評估。 | zh_TW |
dc.description.abstract | Hepatitis B virus (HBV) is a small DNA virus consisting of a nucleocapsid, with a 3.2-kb viral genome, surrounded by an envelope. HBV has infected 2 billion people worldwide, of which 350 million are chronically infected. Chronic HBV infection contributes to severe liver diseases, including cirrhosis and hepatocellular carcinoma. Hepatitis delta virus (HDV) is a satellite virus of HBV as it is coated with the hepatitis B surface antigen (HBsAg) important for the virus infection. HDV has infected 15 million people. Super-infection of chronic HBV patients with HDV highly increases the risk of severe symptoms and accelerates cancer progression. Recently, studies identified sodium taurocholate cotransporting polypeptide (NTCP) as a host receptor for HBV and HDV entry. In this study, a liver-specific human NTCP-expressing in vitro system for HBV infection was established. HBV and HDV infectious particles were prepared in vitro. HBV particles can be stably and continuously produced in HepG2.2.15 cells. While HDV particles were produced by transfecting HepG2.2.15 cells with pSVD2 that contains a dimeric HDV cDNA under the control of the simian virus 40 early promoter. In addition, this study revealed that the presence of HDV increased the level of HBV particles produced by HepG2.2.15 cells. Furthermore, liver-specific expression of exogenous human NTCP conferred the susceptibility in HepG2 cells for HBV infection. The system would provide a convenient platform to study viral infection, virus-virus interaction, pathogenesis, and drug evaluation. | en |
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dc.description.tableofcontents | 目錄
致謝 ..................................................................................................................................................... I 中文摘要 ............................................................................................................................................ II 英文摘要 .......................................................................................................................................... III 縮寫表 ............................................................................................................................................... IV 第一章 緒論 ..................................................................................................................................... 1 1.1病毒性肝炎 ............................................................................................................................... 1 1.2 B型肝炎病毒之發現 ................................................................................................................ 1 1.3 B型肝炎病毒之感染方式與流行病學 .................................................................................... 2 1.4 B型肝炎病毒顆粒組成 ............................................................................................................ 3 1.5 B型肝炎表面抗原 .................................................................................................................... 3 1.6 D型肝炎病毒之發現 ................................................................................................................ 4 1.7 D型肝炎病毒之感染方式與流行病學 .................................................................................... 4 1.8 D型肝炎病毒顆粒組成 ............................................................................................................ 5 1.9 D型肝炎病毒delta抗原 .......................................................................................................... 5 1.10 Delta抗原之結構與功能 ........................................................................................................ 6 1.10.1 Coiled-coil結構 ................................................................................................................ 7 1.10.2核位訊息 ........................................................................................................................... 7 1.10.3核仁素結合位置 ............................................................................................................... 7 1.10.4 Helix-loop-helix區域........................................................................................................ 8 1.10.5細胞核輸出訊號 ............................................................................................................... 8 1.10.6 Isoprenylation motif .......................................................................................................... 9 1.10.7 Clathrin heavy chain binding motif. .................................................................................. 9 1.11 B型與D型肝炎病毒之功能性受體 ................................................................................... 10 第二章 實驗材料來源 .................................................................................................................... 12 2.1大腸桿菌株 .............................................................................................................................. 12 2.2細胞株 ..................................................................................................................................... 12 2.3藥品 ......................................................................................................................................... 12 2.4酵素 ......................................................................................................................................... 15 2.5抗體 ......................................................................................................................................... 15 2.5.1初級抗體 ........................................................................................................................... 15 2.5.2次級抗體 ........................................................................................................................... 16 2.6細胞培養液與試劑 .................................................................................................................. 16 2.7套組試劑 ................................................................................................................................. 17 第三章 實驗方法 ............................................................................................................................ 18 3.1實驗室提供之質體 .................................................................................................................. 18 3.2本研究構築之質體 .................................................................................................................. 19 3.3勝任細胞之製備 ...................................................................................................................... 20 3.4接合反應 ................................................................................................................................. 20 3.5細菌轉型 ................................................................................................................................. 21 3.6質體之小量製備 ...................................................................................................................... 21 3.7質體之中量製備 ...................................................................................................................... 22 3.8 DNA瓊脂膠電泳 .................................................................................................................... 23 3.9細胞株繼代培養 ...................................................................................................................... 24 3.10 DNA轉染 .............................................................................................................................. 25 3.11細胞基因體DNA之收取 ..................................................................................................... 25 3.12細胞RNA之收取 .................................................................................................................. 26 3.13病毒顆粒之收集 .................................................................................................................... 27 3.14 B型肝炎病毒DNA之收取 .................................................................................................. 27 3.15 D型肝炎病毒RNA之收取 .................................................................................................. 28 3.16反轉錄反應 ............................................................................................................................ 28 3.17聚合酶連鎖反應 .................................................................................................................... 28 3.18即時聚合酶連鎖反應 ............................................................................................................ 29 3.19細胞蛋白質之收取 ................................................................................................................ 29 3.20蛋白質定量 ............................................................................................................................ 30 3.21正十二烷硫酸鈉-聚丙烯醯胺膠體電泳 ............................................................................... 30 3.22西方墨點法 ............................................................................................................................ 31 3.22.1半乾式轉印 ..................................................................................................................... 31 3.22.2免疫墨點分析 ................................................................................................................. 32 3.23免疫螢光染色 ........................................................................................................................ 32 3.24病毒感染 ............................................................................................................................... 33 第四章 實驗結果 ............................................................................................................................ 34 4.1以HepG2.2.15細胞株生產HBV病毒顆粒 .......................................................................... 34 4.1.1製備並純化HBV病毒顆粒 ............................................................................................. 34 4.1.2定量HBV病毒顆粒之titers ............................................................................................ 35 4.2以質體pSVD2轉染HepG2.2.15細胞株表現delta抗原以生產HDV病毒顆粒 .............. 35 4.2.1於HepG2.2.15細胞株表現delta抗原 ............................................................................ 35 4.2.2製備並純化HDV病毒顆粒............................................................................................. 36 4.2.3質體pSVD2之轉染增加HepG2.2.15細胞株所釋放之HBV病毒顆粒 ..................... 37 4.3以質體ssAAV-hNTCP-V5-His轉染HepG2細胞株表現人類NTCP蛋白質以建立HBV之感染系統 ....................................................................................................................................... 37 4.3.1質體ssAAv-hNTCP與ssAAV-hNTCP-V5-His之構築 ................................................ 37 4.3.2質體ssAAv-hNTCP-V5-His專一性於HepG2細胞株表現人類NTCP蛋白質 .......... 38 4.3.3表現人類NTCP蛋白質之HepG2細胞株可受到HBV感染 ....................................... 39 第五章 討論 ................................................................................................................................... 40 圖表 ................................................................................................................................................... 44 附錄 ................................................................................................................................................... 56 參考文獻 ........................................................................................................................................... 64 | |
dc.language.iso | zh-TW | |
dc.title | D型肝炎病毒增進B型肝炎病毒之產量 | zh_TW |
dc.title | Hepatitis delta virus enhances the production of hepatitis B virus | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹迺立(Nei-Li Chan),董馨蓮(Sin-Lian Doong),林淑華(Shu-Wha Lin) | |
dc.subject.keyword | B型肝炎病毒,D型肝炎病毒,人類NTCP,病毒感染系統,病毒間交互作用, | zh_TW |
dc.subject.keyword | HBV,HDV,human NTCP,virus infection system,virus-virus interaction, | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-14 | |
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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檔案 | 大小 | 格式 | |
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ntu-103-1.pdf 目前未授權公開取用 | 1.88 MB | Adobe PDF |
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