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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲 | |
dc.contributor.author | Yi-Jiun Lin | en |
dc.contributor.author | 林怡君 | zh_TW |
dc.date.accessioned | 2021-06-15T06:55:51Z | - |
dc.date.available | 2021-12-31 | |
dc.date.copyright | 2011-03-03 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-02-09 | |
dc.identifier.citation | 1. Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiology and Molecular Biology Reviews 64: 51-68.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48413 | - |
dc.description.abstract | B型肝炎病毒在進入宿主後如何造成持續性感染是延宕已久的問題,之前利用以高壓流體力學注射方式活體轉染的小鼠模式來研究與病毒持續性表現或清除有關的病毒因子,已知B型肝炎核心蛋白是造成病毒清除的主要病毒因子,因此本篇論文的主要目的是進ㄧ步地探討其影響病毒清除的詳細機制。首先,利用一系列的核心蛋白突變株,我們發現核心蛋白第176到185個胺基酸片段為導致B型肝炎病毒清除的決定性區域,但是此區域並不包含重要的抗原決定位。當此片段缺失時會延長B型肝炎病毒在小鼠中的表現,但並不影響病毒在肝細胞中的複製、轉錄、與病毒抗原的表現。此外,此片段的缺失也會使小鼠對B型肝炎病毒無法產生適當的體液型及細胞型免疫反應。再者,利用核鞘組裝缺失的核心蛋白突變株,更進ㄧ步揭示核酸核鞘的結構對於核心蛋白導致病毒清除之必要性,同時包含在核酸核鞘中的病毒或是細胞之 RNA是影響免疫反應及病毒清除所必須。綜合上述的研究成果,我們提出了B型肝炎病毒核酸核鞘是B型肝炎病毒的病原相關分子模式的假說,這個病原相關分子模式與病毒清除與否息息相關。本研究對於這個目前仍在發展中的領域注入新的觀念,值得更深入地探究。 | zh_TW |
dc.description.abstract | The mechanism of hepatitis B virus (HBV) persistence following its exposure has long been an unresolved question. Using a hydrodynamics-based mouse model for HBV persistence, we have previously found that HBV core protein (HBc) was the major determinant viral factor for HBV clearance. Therefore, my thesis aimed to further explore the underlying mechanisms. First, we determined the region of HBc protein that is crucial for HBV clearance in mice by using a series of HBcAg deletion mutants. Interestingly, the very C-terminal 10-residue region HBc176-185 of HBc protein, which does not appear to contain a major epitope of T and B cells, contributed to HBV clearance. A deletion of this region prolonged HBV persistence in mice while the viral replication, transcription and translation within hepatocytes remained unaffected. Besides, this particular HBV mutant failed to induce robust humoral and cellular immunity against HBV. Moreover, using an assembly-defective HBV mutant, HBcY132A pAAV/HBV1.2, we revealed the requirement of nucleocapsid structure for inducing effective immunity that leads to HBV clearance in mice. Viral or cellular RNAs, but not DNAs, within the capsid particle were essential for the nucleocapsid to trigger antiviral responses. Finally, these findings prompted us to hypothesize that the HBV nucleocapsid functions as a novel viral pathogen-associated molecular pattern (PAMP) that promotes the viral clearance. This is a burgeoning and exciting research area and may open a new field for future study. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:55:51Z (GMT). No. of bitstreams: 1 ntu-100-D93445005-1.pdf: 1583719 bytes, checksum: 3c0fddb4ae3dc45c691b17925a46346c (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Table of Contents
中文摘要……………………………………………………………………….........…...i Abstract……………………………………………………………………….………...ii Table of contents……………………………………………………………..………...iv Abbreviation…………………………………………………………………………….x Chapter 1: Introduction……………………………………………………………......1 1.1 Life cycle of HBV………………………………………………………………..….1 1.2 Nature history of HBV infection………………………………………….…………2 1.3 HBcAg and the structure of capsid particle………………………………….……....4 1.4 Animal models for the study of HBV persistence…………………………………...5 1.5 The hydrodynamics-based immunocompetent mouse model for HBV persistence....7 1.6 Aim of this study……………………………………………………………………..8 Chapter 2: Materials and Methods…………………………………………………..10 2.1 Plasmid construction…………………………………………….………….….…...10 2.2 Cell line and transfection………………………………………….……………..…10 2.3 Animals and hydrodynamic injection……………………………….………..…….11 2.4 Western blotting…………………………………………………….…………..…..12 2.5 Synthesis of DIG-labeled probe for northern and southern blotting….…………....12 2.6 Preparation of cytoplasmic core-associated DNA……………………….……..…..13 2.7 Isolation of total DNA from a mouse liver…………………….……………..…….13 2.8 RNA extraction and Northern blotting……………………………….………….....14 2.9 Southern blotting…………………………………………………….……..………15 2.10 DNA immunization with in vivo electroporation…………………….…..……….15 2.11 Detection of serum HBV antigens, antibodies, and virions………….………..…..15 2.12 Immunohistochemical staining (IHC)……………………………….………..…..16 2.13 Isolation of mouse splenocytes……………………………………….………..….16 2.14 Enzyme-linked immunospot assay (ELISPOT)……………………….……….....17 2.15 Detection of HBV capsid particles and capsid-associated viral DNA….………...17 Chapter 3: Results…………………………………………………………………….19 3.1 Genetic Dissection of Hepatitis B Virus Core Protein Domains that are Important for HBsAg Persistence in a Hydrodynamics-Based Mouse Model 3.1.1 Validation of the determinant role of HBcAg in the HBV persistence/clearance..19 3.1.2 Carboxyl-terminal domain of HBcAg is critical for HBV clearance…………….20 3.1.3 Deletion of the 10 amino acids at the C-terminus of HBcAg promotes HBV persistence……………………………………………………………………………...23 3.1.4 Impaired immune response to HBV core antigen in mice receiving HBc1–175 pAAV/HBV1.2………………………………………………………………………….24 3.2 Structure Requirement of HBV Core Protein for HBsAg Clearance in a Hydrodynamics-Based Mouse Model 3.2.1 Characterization of a capsid assembly-defective HBc mutant in vitro…………...26 3.2.2 The capsid structure of HBc protein is crucial for the HBV clearance in vivo…...28 3.2.3 Characterization of HBcY132A pAAV/HBV1.2 in vivo…………………………29 3.2.4 Trans-complementation of the intrahepatic HBcY132A protein in the C57BL/6 mice receiving HBeAg/core-null pAAV/HBV1.2……………………………………...30 3.2.5 Defective immune responses to the HBV in the mice receiving HBcY132A pAAV/HBV1.2……………………………………………………………………….…31 Chapter 4: Discussion…………………………………………………………………33 References………………………………………………………………………….…..44 Figures Figure 1. The schematic representation of factors that influence the outcome of HBV infection, either persistent infection or viral clearance, and the immune responses associated with the corresponding outcome……….54 Figure 2. The schematic map of pAAV/HBV1.2……………………….…………..55 Figure 3. The HBcAg expression reduced the HBsAg persistence rate in C57BL/6 mice receiving HBeAg/core-null pAAV/HBV1.2 hydrodynamic injection………………………………………………………………...56 Figure 4. The complementation of HBc expression reduced HBsAg persistent rate in the C57BL/6 mice receiving x-null pAAV/HBV1.2…………………....58 Figure 5. pAAV/HBV1.2 mutants expressing C-terminal-truncated HBcAg enhanced HBsAg persistence in the C57BL/6 mice………………..…..59 Figure 6. The C-terminally truncated pAAV/HBV1.2 mutants promote HBsAg persistence in the BALB/c mice………………………………………..61 Figure 7. HBV replication, transcription, and translation in the C57BL/6 mice receiving WT or mutant pAAV/HBV1.2……………...………………..62 Figure 8. Immunohistochemical staining of the C57BL/6 mice hydrodynamically injected with wild-type and HBc mutant pAAV/HBV1.2……………...64 Figure 9. HBV DNA in the sera of mice receiving wild-type or HBc175 pAAV/HBV1.2………………………………………………………….65 Figure 10. Humoral immune responses and IFN-γ responses stimulated by wild-type or individual mutant forms of HBcAg………………………………….66 Figure 11. The C-terminal 10 amino acid residues of HBcAg did not contain a significant T cell epitope……………………………………………..…67 Figure 12. Capsid formation of different HBc mutants in vitro. HuH-7 cells were transfected with wild-type or mutant pAAV/HBV1.2 as indicated…….68 Figure 13. The viral replication and expression of wild-type and Y132A pAAV/HBV1.2 in vitro…………………………………………………69 Figure 14. The HBcY132A pAAV/HBV1.2 which expressed the capsid assembly-defective HBcAg substantially enhanced the HBsAg and HBeAg persistence rate in C57BL/6 mice……………………………..70 Figure 15. The HBcY132A pAAV/HBV1.2 prominently promoted HBsAg and HBeAg persistence in BALB/c mice……………………………….…..71 Figure 16. Viral replication, transcription and translation in the livers of C57BL/6 mice hydrodynamically injected with wild-type or Y132A pAAV/HBV1.2 at 3 dpi and 38 dpi……………………...…………...…72 Figure 17. Immunohistochemical staining for the intrahepatic expression of HBcAg and HBsAg in the C57BL/6 mice receiving wild-type and Y132A pAAV/HBV1.2……………………………………………..………..….73 Figure 18. Capsid formation of different HBc mutants in vivo……………....….…74 Figure 19. The expression of HBcY132A mutant protein did not reduce the HBsAg persistence rate in the C57BL/6 mice receiving the HBeAg/core-null pAAV/HBV1.2……………………………………………………….…75 Figure 20. Humoral immune responses and IFN-γ responses stimulated by wild-type and Y132A mutant HBcAg………………………………………….….76 Tables Table 1. The sequences of primers used in site-directed mutagenesis for generating various pAAV/HBV1.2 mutants and the resulting mutation site in the HBcAg of each mutant………………………………………..………..78 Table 2. A summary of the phenotypes of wild-type and mutant pAAV/HBV1.2 in vitro and in vivo……………………………………………………..….79 Appendix Figure A1. The effect of pSEAP2-Control, a plasmid used as a transfection efficiency control, on the HBsAg persistence in the mice receiving x-null pAAV/HBV1.2……………………………………………………….…81 | |
dc.language.iso | en | |
dc.title | 以高壓流體注射法建立之小鼠模式探討B型肝炎病毒核心蛋白影響B型肝炎病毒清除之機制 | zh_TW |
dc.title | Characterization of the role of hepatitis B virus core protein in the viral clearance in a hydrodynamics-based mouse model | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 施嘉和,楊宏志,許秉寧,廖楓 | |
dc.subject.keyword | B型肝炎病毒,高壓流體注射,核心蛋白,核酸核鞘,表面抗原血症,病原相關分子模式, | zh_TW |
dc.subject.keyword | Hepatitis B virus (HBV),hydrodynamic injection,HBV core protein/antigen (HBc),nucleocapsid,surface antigenemia,pathogen-associated molecular pattern (PAMP), | en |
dc.relation.page | 81 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-02-09 | |
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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