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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陶秘華 | |
| dc.contributor.author | Pei-Ling Wu | en |
| dc.contributor.author | 吳佩玲 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:34:50Z | - |
| dc.date.available | 2009-08-04 | |
| dc.date.copyright | 2006-08-04 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32168 | - |
| dc.description.abstract | 當今全世界約有三億五千萬人患有慢性B型肝炎，由慢性B型肝炎所引發之肝硬化及肝癌等相關疾病，每年造成的全球死亡人數高達一百多萬，嚴重危害人類的健康。目前治療慢性B型肝炎藥物有免疫調節劑及抗病毒藥物兩類，它們在臨床上雖皆具一定療效，但前者會爲病患帶來諸多不適的副作用，而後者則面臨著引起病毒抗藥性的挑戰。因此，研發對抗B型肝炎病毒的新型藥物，遂成為刻不容緩的研究課題。
基於與抗原間的結合極具專一性及親和力，單株抗體素被廣泛應用於疾病的診斷，近年來更逐漸成為新藥研發的主流之一。過去，抗體藥物的研發方向多著重於調控細胞外蛋白質之生物功能，而涵蓋多數致病因子的細胞內蛋白質反被忽略。隨著抗體工程技術的進展，一個原應分泌至細胞外的抗體分子，經過適當修飾後，可以停留在細胞內部與細胞內抗原結合，進而干擾抗原之正常生理作用。有鑑於此，本研究欲探討此類細胞內抗體（intrabody）作為治療慢性B型肝炎之藥物的可行性。在諸多B型肝炎病毒之病毒蛋白中，大型表面蛋白（LHBsAg）對病毒之複製有舉足輕重的影響，故本研究選其作為細胞內抗體之抗原標的。 過去，我們已將一個能有效辨認大型表面蛋白preS1區域之單株抗體—MA18/7—轉成單鏈抗體型態（single-chain Fv）。本研究中，我們證實該表現質體轉染進入細胞後確能順利表現出MA18/7scFv蛋白質，以作為細胞內抗體。接著，我們分別在兩種B型肝炎病毒體外複製之模式系統中檢驗MA18/7細胞內抗體對病毒複製的影響：一、將病毒基因組DNA與細胞內抗體表現質體DNA共同轉染入肝細胞株；二、以腺病毒載體攜帶病毒基因組DNA進入已穩定表現細胞內抗體之肝細胞株。初步結果發現：在系統一中，MA18/7細胞內抗體對B型肝炎病毒表面蛋白（HBsAg）的分泌量無顯著影響。至於在系統二中，MA18/7細胞內抗體卻能明顯抑制肝細胞分泌B型肝炎病毒大型表面蛋白，然而B型肝炎病毒DNA的總釋放量，以及子代病毒外套膜之存在與否皆不因MA18/7細胞內抗體有所改變。造成此間差異之原因究竟為何，尚需後續實驗加以驗證。 此外，本研究亦計畫修飾MA18/7細胞內抗體之蛋白組成，使之專一地分佈於細胞內特定胞器中，以期提高其尋找目標抗原的成功率，進而加強其抑制能力。目前我們已完成了分布在細胞核或細胞質中的兩種MA18/7細胞內抗體的構築，並藉由免疫螢光染色法證實其分佈位置確如預期。 最後，本研究更進一步探討前述抑制作用之機制。我們分別利用螢光蛋白標籤以及免疫螢光染色法兩種方法證實，MA18/7細胞內抗體與其目標抗原—大型表面蛋白—在細胞內的分佈位置相疊合，初步證明兩者之間可能存有交互作用。 綜合而言，本研究初步發現，抗B型肝炎病毒大型表面蛋白之MA18/7細胞內抗體在蛋白質層次上對病毒有顯著抑制效果。另一方面，雖然抗體與抗原間之交互作用已被證實，惟其詳盡機制尚待日後研究方能解答。不論答案為何，以細胞內抗體作為治療慢性B型肝炎藥物確為值得發展的方向。 | zh_TW |
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| dc.description.tableofcontents | 摘 要 i
ABSTRACT iii ACKNOWLEDGEMENT v TABLE OF CONTENTS vi TABLE OF FIGURES xi TABLE OF ABBREVIATIONS xiii INTRODUCTION 1 1.1 Introduction to hepatitis B virus (HBV) 1 1.1.1 Classification and structure 1 1.1.2 Viral genes and proteins 2 1.1.3 Viral replication cycle 3 1.1.4 L protein: functions and dual transmembrane topology 4 1.2 Pathogenesis of hepatitis B 5 1.3 Current antiviral therapy for chronic hepatitis B (CHB) infection 5 1.4 Antiviral targets of future therapeutic agents 8 1.5 Introduction to intracellular antibody (intrabody) 9 1.5.1 What is an intrabody? 9 1.5.2 Brief history of intrabody 9 1.5.3 Current format of intrabody: single-chain Fv (scFv) 10 1.5.4 Subcellular targeting of intrabody 10 1.5.5 Subcellular modes of intrabody action 11 1.5.6 Applications of intrabody in disease: viral and non-viral . 11 1.5.7 Advantages of intrabody over other intracellular targeting strategies 13 1.6 Promise of intrabody as potential therapeutic agent for CHB 14 1.7 Specific aim & Experimental design 15 MATERIALS AND METHODS 17 2.1 Construction of plasmids 17 2.1.1 Preparation of plasmids 17 2.1.2 Polymerase chain reaction (PCR) 18 2.1.3 1% agarose gel electrophoresis 22 2.1.4 Purification of DNA from agarose gel 22 2.1.5 Zero Blunt TOPO PCR Cloning 23 2.1.6 Restriction enzyme digestion 23 2.1.7 DNA ligation 24 2.1.8 Transformation of bacteria by electroporation 24 2.2 Cell lines and cell culture 25 2.3 Protein expression in eukaryotic cells 26 2.3.1 Transfection with cationic lipid reagents 26 2.3.2 Western blotting 26 2.3.3 Flow cytometry 27 2.3.4 Immunofluorescence staining & confocal microscopy 28 2.4 Preparation of recombinant adenoviral vectors 28 2.4.1 Large-scale amplification and cesium chloride gradient purification 28 2.4.2 Viral particle titration by plaque assay 29 2.5 Adenoviral transduction 30 2.6 Assays detecting HBV viral antigens and DNA 30 2.6.1 ELISA analysis of secretory HBV proteins (HBsAg and LHBsAg) 30 2.6.2 Western blot analysis of intracellular HBV proteins (LHBsAg) 32 2.6.3 Q-PCR analysis of extracellular HBV DNA 32 2.6.4 Southern blot analysis of extracellular HBV DNA 33 RESULTS 35 I. Inhibitory effect of anti-preS1 intrabody on HBV replication 35 3.1 Expression of MA18/7scFv intrabody from p3224-3/MA18/7scFv 35 3.1.1 Protein structure of anti-preS1 MA18/7scFv 35 3.1.2 Expression and subcellular localization of MA18/7scFv intrabody 36 3.2 Plasmid-mediated HBV genome delivery into hepatoma cell lines 38 3.2.1 Genome structure of HBV1.3 38 3.2.2 Secretion of HBsAg from different hepatoma cell lines upon pHBV1.3 transient transfection 39 3.3 Insignificant effect of transiently-expressed MA18/7scFv intrabody on HBsAg secretion caused by pHBV1.3 transfection 39 3.4 Expression of membrane-bound form of MA18/7scFv intrabody in stable HepG2/MA18/7scFv-TM cell 41 3.4.1 Protein structure of membrane-bound MA18/7scFv 41 3.4.2 Surface expression and subcellular localization of membrane-bound form of MA18/7scFv intrabody in HepG2/MA18/7scFv-TM 42 3.5 Adenovirus-mediated HBV genome delivery into hepatoma cell lines 44 3.5.1 Genome structure of Ad/HBV 44 3.5.2 Large-scale production and purification of Ad/HBV 45 3.5.3 Characterization and optimization of Ad/HBV infection on HepG2 45 3.5.4 Ad/HBV-mediated production of LHBsAg from HepG2 cells 46 3.6 Inhibition effect of permanently-expressed MA18/7scFv intrabody on HBV replication caused by Ad/HBV transduction 47 3.6.1 Inhibition effect on LHBsAg secretion 48 3.6.2 Insignificant effect on HBV DNA secretion 48 3.6.3 Insignificant effect on secretory HBV DNA pattern and structure of progeny HBV virions 49 3.7 Construction of cytosol- and nucleus-targeting MA18/7scFv intrabodies 50 3.7.1 Protein structure of MA18/7scFv and MA18/7scFv-NLS 51 3.7.2 Construction of pLNCX/MA18/7scFv and pLNCX/MA18/7scFv-NLS 52 3.7.3 Expression and subcellular localization of cytosol- and nucleus-targeting MA18/7scFv intrabodies in 3T3 cells 53 II. Mechanism underlying the inhibition: Investigating the association between intrabody and antigen 54 3.8 Construction of MA18/7scFv intrabody-EGFP fusion protein 55 3.8.1 Protein structure of MA18/7scFv-EGFP fusion protein 55 3.8.2 Construction of p3224-3/MA18/7scFv-EGFP expression plasmid 55 3.8.3 Expression and subcellular localization of MA18/7scFv-EGFP fusion protein in 3T3 cells 56 3.9 Construction of two kinds of preS1 Ag-DsRed2 fusion proteins 58 3.9.1 Protein structure of preS1(10-38)-DsRed2 and preS1-DsRed2 fusion proteins 59 3.9.2 Construction of ppreS1(10-38)-DsRed2 and ppreS1-DsRed2 expression plasmids 59 3.9.3 Subcellular localizations of preS1(10-38)-DsRed2 and preS1-DsRed2 fusion proteins in 3T3 cells 60 3.10 Co-localization of MA18/7scFv-EGFP and Ag-DsRed2 in 3T3 cells 61 3.11. Co-localization of MA18/7scFv intrabody and LHBsAg in HepG2 cells 62 DISCUSSION 65 4.1 Aggregation of cytosolic MA18/7scFv intrabody 65 4.2 Absence of inhibition effect of MA18/7scFv intrabody in plasmid-mediated HBV replication model 66 4.3 Inhibition effect of MA18/7scFv intrabody in adenovirus-mediated HBV replication model 67 4.4 Effect of MA18/7scFv intrabody on the structure of progeny virions 68 4.5 Prospect and future work 69 REFERENCES 70 FIGURES 78 APPENDIX A Primers 116 APPENDIX B Molecular Biology Reagents 117 | |
| dc.language.iso | en | |
| dc.subject | 單鏈抗體 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | B型肝炎 | zh_TW |
| dc.subject | 慢性B型肝炎感染 | zh_TW |
| dc.subject | 病毒複製 | zh_TW |
| dc.subject | 抗病毒 | zh_TW |
| dc.subject | 抗病毒藥物 | zh_TW |
| dc.subject | 療法 | zh_TW |
| dc.subject | 治療 | zh_TW |
| dc.subject | 藥物開發 | zh_TW |
| dc.subject | 抗體 | zh_TW |
| dc.subject | 抗原 | zh_TW |
| dc.subject | 細胞內抗體 | zh_TW |
| dc.subject | 胞內抗體 | zh_TW |
| dc.subject | 表面抗原 | zh_TW |
| dc.subject | 大型表面蛋白 | zh_TW |
| dc.subject | 單鏈 | zh_TW |
| dc.subject | antigen | en |
| dc.subject | HBV | en |
| dc.subject | chronic hepatits B | en |
| dc.subject | CHB | en |
| dc.subject | viral replication | en |
| dc.subject | antiviral | en |
| dc.subject | antiviral agent | en |
| dc.subject | therapy | en |
| dc.subject | drug development | en |
| dc.subject | antibody | en |
| dc.subject | hepatitis B virus | en |
| dc.subject | viral antigen | en |
| dc.subject | intracellular antibody | en |
| dc.subject | intrabody | en |
| dc.subject | surface antigen | en |
| dc.subject | large surface antigen | en |
| dc.subject | LHBsAg | en |
| dc.subject | HBsAg | en |
| dc.subject | preS1 | en |
| dc.subject | MA18/7 | en |
| dc.subject | single-chain | en |
| dc.subject | variable domain | en |
| dc.subject | single-chain variable domain | en |
| dc.subject | scFv | en |
| dc.title | 發展抗B型肝炎病毒大型表面蛋白之細胞內抗體作為治療慢性B型肝炎的藥物 | zh_TW |
| dc.title | Intrabody directed against HBV LHBsAg as potential therapeutic agents for CHB | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃麗華,吳慧琳,葉秀慧 | |
| dc.subject.keyword | B型肝炎病毒,B型肝炎,慢性B型肝炎感染,病毒複製,抗病毒,抗病毒藥物,療法,治療,藥物開發,抗體,抗原,細胞內抗體,胞內抗體,表面抗原,大型表面蛋白,單鏈,單鏈抗體, | zh_TW |
| dc.subject.keyword | hepatitis B virus,HBV,chronic hepatits B,CHB,viral replication,antiviral,antiviral agent,therapy,drug development,antibody,antigen,viral antigen,intracellular antibody,intrabody,surface antigen,large surface antigen,LHBsAg,HBsAg,preS1,MA18/7,single-chain,variable domain,single-chain variable domain,scFv, | en |
| dc.relation.page | 117 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-27 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 未授權公開取用 | 1.78 MB | Adobe PDF |
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