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研究生:唐嘉輿
研究生(外文):Jia Yu Tang
論文名稱:調控不同台灣第二基因型D型肝炎病毒株間複製力強弱的分子機轉
論文名稱(外文):Regulation of the replication activity of Taiwanese hepatitis delta virus genotype 2
指導教授:趙玫趙玫引用關係
指導教授(外文):M. Chao
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:141
中文關鍵詞:D型肝炎病毒核酶D型抗原啟動子
外文關鍵詞:hepatitis delta virusribozymesS-HDAgpromoter
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目錄
國家圖書館博碩士學位論文延後公開申請書
指導教授推薦書
論文口試委員審定書
致謝 iii
摘要 iv
Abstract vi
目錄 viii
圖目錄 x
表目錄 xi
附錄目錄 xii
第一章 前言 - 1 -
1.1 D型肝炎 - 1 -
1.2 D型肝炎病毒的構造 - 2 -
1.3 D型肝炎病毒的基因體 - 2 -
1.4 D型肝炎抗原 - 6 -
1.5 HDV基因體的複製 - 9 -
1.6 HDV基因型 - 13 -
1.7 HDV不同基因型的差異 - 15 -
1.8實驗室初步結果 - 17 -
1.8.1 HDV-2 replication clones - 17 -
1.8.2 Replication levels - 18 -
1.8.3 Transactivation of S-HDAg - 18 -
第二章 研究動機與方向 - 20 -
2.1 Flowchart - 20 -
2.2 研究動機 - 20 -
第三章 材料與方法 - 22 -
3.1 細菌培養 - 22 -
3.2 大腸桿菌的轉型作用 - 22 -
3.3 Mini質體DNA的萃取 - 22 -
3.4 Midi質體DNA的萃取 - 23 -
3.5細胞培養 - 24 -
3.6 DNA轉染 - 24 -
3.7細胞內RNA萃取 - 25 -
3.8細胞內 protein萃取 - 25 -
3.9北方墨點法(Northern blot) - 26 -
3.10聚丙醯氨凝膠電泳(SDS-PAGE) - 27 -
3.11西方墨點法(Western blot) - 28 -
3.12 Site-directed mutagenesis - 30 -
3.13 傳統聚合酵素連鎖反應 (PCR) - 31 -
3.14 Overlapping extension (OE)-PCR - 32 -
3.15質體的建構 - 33 -
第四章 結果 - 41 -
4.1探討HDV-2HDAg trans-activation activity - 41 -
4.1.1 N15 HDAg支持複製的功能性分析 - 41 -
4.1.2 N14HDAg支持複製的功能性分析 - 42 -
4.2探討HDV-2的cis-elements對於複製的能力 - 43 -
4.2.1改變HDV-2左邊區域桿狀結構對於複製的影響 - 43 -
4.2.2探討HDV-2的ribozyme 區域內的突變對於複製的影響 - 51 -
第五章 結論 - 54 -
5.1 探討HDV-2 HDAg trans-activation activity - 54 -
5.2 探討HDV-2的cis-elements對於複製的能力 - 56 -
5.2.1 HDV-2 左邊桿狀結構的改變對於複製的影響 - 56 -
5.2.2 Ribozyme mutation影響複製的分析 - 57 -
參考文獻 - 60 -
圖 - 67 -
表 - 91 -
附錄 - 103 -

圖目錄
圖 一 HDV-2 N14的3個replication clones序列比對 - 67 -
圖 二 N1、N2 chimera的複製能力 - 68 -
圖 三 N1 Loop mutant左邊桿狀結構預測圖 - 69 -
圖 四 N1 Loop mutagenesis的複製力 - 70 -
圖 五 N1、N15 HDAg胺基酸序列比對圖 - 71 -
圖 六 N14-11、N15 HDAg胺基酸序列比對圖 - 72 -
圖 七 N1、N15 chimeric HDAg支持第二基因型的複製力 - 73 -
圖 八 N1、N15 chimeric HDAg支持D2M的複製力 - 74 -
圖 九 N14、N15 chimeric HDAg支持D2M的複製力 - 75 -
圖 十 N14、N15 chimeric HDAg支持D2N14M的複製力 - 76 -
圖 十一 測試D1.2N1 replication clone的複製能力 - 77 -
圖 十二 N1左邊桿狀結構預測圖 - 78 -
圖 十三 N1左邊桿狀mutants的結構圖和複製能力 - 79 -
圖 十四 N1 左邊桿狀 mutants結構圖和複製力 - 80 -
圖 十五 N1左邊桿狀mutant結構的圖 - 82 -
圖 十六 HDV-2 N14的3個replication clones序列比對圖 - 83 -
圖 十七 HDV-1(T)和HDV-2 (N1、N2)的genomic、antigenomic ribozyme序列比對 - 84 -
圖 十八 T、N1和N2ribozyme基因股和反基因股的二級結構 - 85 -
圖 十九 N1 ribozyme基因股和反基因股的二級結構 - 86 -
圖 二十 N1 ribozyme mutagenesis的複製力 - 87 -
圖 二十一 比對實驗室HDV-2、HDV-1(T)的ribozyme序列 - 88 -
圖 二十二 N1、N15 HDAg胺基酸序列比對圖 - 89 -
圖 二十三 N14、N15 HDAg胺基酸序列比對圖 - 90 -

表目錄
表 一 HDV-2左邊區域桿狀結構圖 - 91 -
表 二 建構N1左邊桿狀結構之相關primer - 93 -
表 三 建構ribozyme mutagenesis之相關primer - 95 -
表 四 建構Chimeric S-HDAg之相關primer - 96 -
表 五 建構point mutation之相關PCR反應條件 - 97 -
表 六 Chimeric S-HDAg之相關PCR反應條件 - 99 -

附錄目錄
附錄 一 HDV的anti-genomic(αG)RNA - 103 -
附錄 二 HDV不分枝桿結構(unbranched rod-like structure) - 104 -
附錄 三 HDV ribozyme基因股和反基因股的二級結構 - 105 -
附錄 四 HDV桿狀結構圖 - 106 -
附錄 五 HDV基因體的複製和HDAg的產生 - 107 -
附錄 六 HDV基因體的複製機制 - 108 -
附錄 七 HDV-1的promoter桿狀結構圖 - 109 -
附錄 八 實驗室HDV-2 的7個replication clones複製能力圖 - 110 -
附錄 九 由高雄長庚李全謨醫師提供病人檢體N1、N2的資訊 - 111 -
附錄 十 不同HDV-2的S-HDAg支持D2M的複製 - 112 -
附錄 十一 不同HDV-2的S-HDAg支持D2N1M的複製 - 113 -
附錄 十二 不同HDV-2的S-HDAg支持D2N14-11M的複製 - 114 -
附錄 十三 pCR3.1-D2N1 - 115 -
附錄 十四 pCR3.1-D1.2N1 - 117 -
附錄 十五 N1左邊桿狀結構突變的質體 - 118 -
附錄 十六 ribozyme mutangensis的質體 - 119 -
附錄 十七 實驗室七個HDV-2核苷酸與胺基酸的序列同源性比對 - 121 -
附錄 十八 HDV-2 GenBank中19個clones左邊桿狀結構原始圖 - 122 -
附錄 十九 N1 Loop mutagenesis的複製力(repeat data) - 123 -
附錄 二十 N1 ribozyme mutagenesis的複製力(repeat data) - 124 -
附錄 二十一 N1 ribozyme mutagenesis的複製力(repeat data) - 125 -
附錄 二十二 Ribozyme區域突變的桿狀結構圖 - 126 -
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