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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊宏志 | |
| dc.contributor.author | Yu-Hsiang Chen | en |
| dc.contributor.author | 陳煜翔 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:35:26Z | - |
| dc.date.available | 2018-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49572 | - |
| dc.description.abstract | B型肝炎病毒感染會造成肝硬化與肝癌。雖然已有有效之疫苗可以預防感染，目前全世界仍有2.4億人帶有慢性B型肝炎病毒感染，且每年約有78萬人死於B型肝炎相關併發症。日前治療上主要使用的藥物為核苷類似物與干擾素兩大類，然而這兩種藥物只能抑制病程的進行以及癌症發生率而不能完全清除病毒，原因為此藥物無法針對病毒之共價閉合環狀DNA (covalently closed circular DNA, cccDNA) 進行降解。病毒之cccDNA被發現會於細胞內穩定存在，且此DNA為病毒轉錄之模板，其會轉錄出病毒的RNA。病毒會利用pregenomic RNA (pgRNA) 進行反轉錄而完成複製，故只要cccDNA仍存在於細胞中，病毒就有可能伺機而起。cccDNA由病毒之relaxed circular DNA (rcDNA) 轉變而來，科學家認為此過程需由宿主的DNA修復蛋白協助，然而目前尚不清楚哪些宿主蛋白參與cccDNA形成或其穩定。為了發展興新藥物以及了解B型肝炎病毒學，我們必須更了解此過程。為此，目前已有許多科學家嘗試將病毒做成載體，攜帶著外來基因作為報告基因，希望藉由高通量篩選的方式了解病毒複製過程中參與的宿主基因或開發藥物。然而目前所建立之報告系統多用於陣列篩選 (Arrayed screening) 。另一方面，能用來做匯集篩選 (Pooled screening) 的載體其感染的效率並未被分析，且其重組病毒之複製能力令人質疑。為了以Pooled screening進行全基因組的分析，進而了解參與病毒複製過程中的宿主基因，本研究建立了攜帶著抗藥性基因與小綠色螢光蛋白的重組病毒載體，並分析此載體於細胞複製之能力、產生病毒之能力以及報告基因之表現量。最後，我們也建立一株表達B型肝炎病毒核心抗原的細胞，並分析重組病毒感染此細胞之能力。 | zh_TW |
| dc.description.abstract | Hepatitis B virus (HBV) is the causative agent of chronic hepatitis B, which often leads to liver cirrhosis and hepatocellular carcinoma. Although current vaccines can prevent the infection of HBV, there are 240 million chronic HBV carriers worldwide and more than 780,000 people decease every year due to the complications. Current treatment for chronic HBV infection includes nucleos(t)ide analogues (NAs) and interferon-alpha (IFN-α). However, current therapies only retard the progression of disease and reduce the incidence of liver cancer but do not cure hepatitis B infection due to their little effect on viral covalently closed circular DNA (cccDNA) stability. It has been shown that cccDNA is stable in non-dividing hepatocytes after infection. As the viral transcriptional template, cccDNA is transcribed into viral pregenomic RNA (pgRNA) and subgenomic RNAs (sgRNAs). The pgRNA is subsequently reversely transcribed into viral relaxed circular DNA (rcDNA) to complete the viral replication cycle. Hence, the persistence of cccDNA is considered the main reason for viral relapse after drug withdraw. It is generally assumed that cccDNA is converted from rcDNA through the host repair machinery. However, the detailed mechanisms and host factors participating in this process remain unknown. A variety of recombinant viral vectors have been developed in hopes of unraveling the process. However, most existing HBV reporter systems were designed for arrayed screening; other recombinant viral vectors which can be used to perform pooled screening lack the assessment of the infectivity. In order to identify host genes involved in HBV replication through the genome-wide screening, we created recombinant HBV vectors carrying blasticidin-S-deaminase, conferring resistance to Blasticidin S, and iLOV, a FMN-binding fluorescent protein. We proved that our recombinant HBV reporter systems were functional in the replication, virion production and cargo gene expression in human hepatoma cell lines. Besides, we also established a cell line which expresses the HBV core protein, and evaluated the infectivity of the virus for this cell line. Although the infectivity of the virus still requires further investigation, the recombinant vectors will be a robust tool to study HBV replication as long as the infectivity is finally confirmed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:35:26Z (GMT). No. of bitstreams: 1 ntu-105-R03445109-1.pdf: 1986594 bytes, checksum: e4e7eca736d934fd10914ebe54436780 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 致謝 IV
中文摘要 VI ABSTRACT VIII 1. INTRODUCTION 1 1.1 Classification of Hepatitis B Virus 1 1.2 Virion Structure and Viral Proteins of HBV 1 1.3 Viral Replication Cycle 3 1.4 Current Treatment and Persistence of HBV Infection 4 1.5 The Studies of cccDNA 4 1.6 Challenges to Study HBV Replication and A New Culture Model for HBV Infection 6 1.7 Current HBV Reporter Systems 7 1.7.1 cccDNA reporter systems in HBV-expressing cell lines 7 1.7.2 Recombinant hepatitis B virus vectors used to study HBV infection 9 2. SPECIFIC AIMS 12 3. MATERIALS AND METHODS 13 3.1 Cell Lines and Cell Culture 13 3.2 Plasmid constructs 13 3.3 DNA Transfection 14 3.4 Establishment of Core-expressing HepG2-NTCP-Core Cell Line 15 3.5 DIG-labeled Probe Synthesis 16 3.6 Extraction of Viral Nucleic Acid 16 3.7 Southern Blot Analysis 18 3.8 Northern Blot Analysis 19 3.9 Western Blot Analysis 20 3.10 Preparation of Recombinant HBV Virions 21 3.11 Native Agarose Gel Analysis of HBV Virions 22 3.12 Quantification of Extracellular HBV DNA by qPCR 22 3.13 HBV Infection 23 3.14 Immunofluorescence Assay 23 4. RESULTS 24 4.1 Construction of Recombinant HBV Vectors 24 4.2 Establishment of Core-expressing Cell Line 24 4.3 Evaluation of RNA and Cargo Genes Expression from rHBV Vectors 25 4.4 Assessment of rHBV Replication Efficiency 26 4.5 Characterization of Virion Production by rHBV 26 4.6 Infection of HepG2-NTCP-Core with rHBV Vectors 28 5. DISCUSSION 29 6. FIGURES 34 Figure 1. Schematic representation of constructs used to produce virus. 34 Figure 2. Establishment of cell lines expressing HBV core in a tetracycline-inducible manner. 35 Figure 3. Analysis of RNA expression driven by the authentic HBV promoter from the recombinant HBV vectors. 36 Figure 4. The reporter gene expression from recombinant HBV pgRNA driven by the CMV promoter. 38 Figure 5. Replication efficiency of the recombinant HBV vectors. 39 Figure 6. Production of the recombinant virions by trans-complementation. 41 Figure 7. Infection of HepG2-NTCP-Core cells with recombinant HBV. 42 REFERENCES 43 SUPPREMENTARY INFORMATION 48 Supplementary figure 1. Establishment of reporter systems based on transfection 49 | |
| dc.language.iso | en | |
| dc.subject | 重組病毒 | zh_TW |
| dc.subject | 報告系統 | zh_TW |
| dc.subject | 高通量篩選 | zh_TW |
| dc.subject | B 型肝炎病毒 | zh_TW |
| dc.subject | cccDNA | zh_TW |
| dc.subject | Pooled library screening | en |
| dc.subject | Hepatitis B virus | en |
| dc.subject | Reporter system | en |
| dc.subject | cccDNA | en |
| dc.subject | Recombinant viral vector | en |
| dc.title | 建立B型肝炎病毒感染之報告系統 | zh_TW |
| dc.title | To Establish a Reporter System for Hepatitis B Virus Infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳培哲,董馨蓮 | |
| dc.subject.keyword | B 型肝炎病毒,cccDNA,高通量篩選,報告系統,重組病毒, | zh_TW |
| dc.subject.keyword | Hepatitis B virus,cccDNA,Reporter system,Pooled library screening,Recombinant viral vector, | en |
| dc.relation.page | 49 | |
| dc.identifier.doi | 10.6342/NTU201602590 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-17 | |
| 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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