臺灣博碩士論文加值系統

當冠狀病毒感染細胞時，一系列長短不一的次級基因體（subgenomic RNA , sgmRNA）會形成，以轉譯成各種結構蛋白，這些蛋白質會與病毒顆粒的形成及致病機轉有關。之前的研究已知，正股次級基因體有能力以自己為模板形成負股的次級基因體，而在正股次級基因體上的特殊結構，是否會影響負股次級基因體的形成，目前仍屬未知。在本實驗，我們構築冠狀病毒N蛋白的次級基因體，並將其特殊結構分別刪除而形成一系列突變株，最後利用即時定量聚合酶連鎖反應比較負股次級基因體的生成效率，以研究正股次級基因體上有那些特殊結構，為負股次級基因體形成所需之cis-acting elements。由實驗結果可知：（1）將正股次級基因體5’端的領導序列（leader sequence）刪除，會嚴重抑制負股次級基因體的形成；（2）刪除正股次級基因體其3’端的bulged stem-loop（BSL）不會影響負股次級基因體的形成，但若將pseudoknot（PK）結構刪除，則會使負股次級基因體的形成量大幅增加；（3）正股次級基因體其3’端倒數第15至第34個核苷酸，對於負股次級基因體的形成很重要；（4）正股次級基因體3’端最後一個核苷酸的種類，也會影響負股次級基因體的形成。由以上結果可知，正股次級基因體的3’端及5’端均含有對於負股次級基因體形成所需之cis-acting elements。

During coronavirus infection, a nested set of subgenomic mRNAs (sgmRNAs) are made from which structural and accessory proteins are expressed. These proteins are associated with viral assembly and pathogenesis. It has been demonstrated that sgmRNAs is able to serve as a template for synthesis of its negative-strand [(-)-strand] complement. The cis-acting elements on positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis, however, have not been identified. Here, we employed head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) to analyze cis-acting element requirements on bovine coronavirus (BCoV) sgmRNA 7 for synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows: (1) deletions of 5’-terminal leader sequence on sgmRNA7 blocked the synthesis of (-)-strand sgmRNA complement, (2) deletions of buldged stem-loop had no effect on (-)-strand sgmRNA synthesis; however, deletion of pseudoknot increased the yield of (-)-strand sgmRNA, (3) nucleotides positioned from -15 to 34 of the 3’-terminal region on sgmRNA 7 are required for efficient (-)-strand sgmRNA synthesis, (4) nucleotide species at the 3’-most position (-1) on sgmRNA 7 is pivotal for synthesis of sgmRNA 7 (-)-strand. These results together suggest, in principle, the 5’- and 3’-terminal sequences of sgmRNA are critical for efficient (-)-strand sgmRNA synthesis.

摘要 i
Abstract ii
目次 iii
表次 vi
圖次 vii
第一章 前言 1
第二章 文獻探討 2
第一節 冠狀病毒之基本簡介 2
1.1 冠狀病毒之背景 2
1.2 冠狀病毒基因體及次級基因體之結構 2
第二節 冠狀病毒之複製及轉錄 3
2.1 冠狀病毒之生活史 3
2.2 冠狀病毒RNA基因體之複製 3
2.3 冠狀病毒RNA次級基因體之生成 4
第三節 冠狀病毒之cis-acting elements 5
3.1 cis-acting elements的特性 5
3.2 冠狀病毒基因體之cis-acting elements 5
3.3 冠狀病毒次級基因體之cis-acting elements 6
第四節 利用冠狀病毒的DI-RNA系統研究cis-acting elements 6
4.1 牛冠狀病毒天然缺陷株RNA 6
4.2 以DI-RNA做為cis-acting elements之研究模組 6
4.3 研究病毒負股RNA生合成所遭遇之困難 7
第三章 材料與方法 8
第一節 野外株與突變株次級基因體質體之構築 8
1.1 次級基因體野外株質體之構築 8
1.2 利用重疊PCR法構築突變株 8
1.3 利用XL-TOPO® Vector （invitrogenTM）系統將突變之PCR DNA片段進行選殖 9
1.4 利用PCR篩選內含突變序列的菌落 10
1.5 質體DNA之萃取 10
1.6 利用限制酵素切割突變DNA片段 11
1.7 DNA片段純化 11
1.8 將突變DNA片段接入pGEM®-3Zf（-）載體 11
1.9 利用100%酒精沉澱DNA連接反應之產物 12
1.10 突變DNA片段之轉型作用及選殖 12
1.11 利用PCR篩選內含突變序列的菌株 12
1.12 質體DNA之萃取 13
第二節 體外轉錄反應及定量 13
2.1 利用單一限制酵素Mlu I使環形質體形成線狀 13
2.2 體外轉錄反應（In vitro transcription） 13
2.3 RNA純化 14
2.4 RNA濃度測定 14
第三節 野外株及突變株RNA之轉染 14
3.1 細胞及病毒 14
3.2 牛冠狀病毒DI-RNA及sgmRNA之轉染 15
第四節 利用即時定量聚合酶連鎖反應偵測BCoV負股DI-RNA之生成 15
4.1 自HRT-18細胞萃取RNA 15
4.2 去除RNA 5’端之磷酸根端帽 16
4.3 利用T4 RNA LigaseⅠ將RNA 3，端與5，端頭尾相連 16
4.4 互補DNA（cDNA）之合成 16
4.5 即時定量聚合酶連鎖反應 17
第五節 以北方墨漬法偵測BCoV正股DI-RNA、正股M次級基因體及18S rRNA之生成 17
5.1 變性瓊脂凝膠（denaturing agarose gel）及北方雜交緩衝液製備 17
5.2 探針製備 17
5.3 電泳分析 18
5.4 RNA轉漬及固定 18
5.5 雜合反應（Hybridization） 18
5.6 X光片呈色 18
第六節 統計分析 18
第四章 實驗結果 19
第一節 冠狀病毒DI-RNA 5’端由領導序列至N蛋白基因序列之間的421個核苷酸對於負股次級基因體生成的重要性 19
第二節 正股次級基因體的cis-acting elements對於負股次基因體製造之重要性 19
第三節 確認正股次級基因體3’ UTR最後55個核苷酸中，對於負股次級基因體製造所需之cis-acting elements 20
第四節 冠狀病毒3’ UTR最後一個核苷酸的種類對於負股次基因體形成的重要性 20
第五節 正股次級基因體中領導序列及其特殊結構對於負股基因體生成的重要性 21
第五章 討論 38
參考文獻 40

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