臺灣博碩士論文加值系統

茲卡病毒屬於黃病毒科黃病毒屬，在1947年由非洲烏干達被分離出來，近幾年茲卡病毒在中南美洲與東南亞等地傳出大規模的疫情，同時許多被茲卡病毒感染的孕婦產下的胎兒有小頭症的案例傳出，因而受到重視。過去文獻提到許多黃病毒NS1蛋白與病毒複製有關，近幾年的研究指出黃病毒NS1蛋白除了病毒複製之外，還會在感染初期釋放至細胞外干擾宿主體內的免疫系統與協助病毒對宿主的感染，綜上所述茲卡病毒的NS1蛋白應該也有協助病毒複製或干擾宿主免疫系統等功能，為此我們決定先觀察茲卡病毒的RNA複製與NS1蛋白，再接著過量表現NS1蛋白觀察對病毒生活史的影響；除此之外，有許多的文獻顯示分泌至細胞外的NS1蛋白並非以單倍體的形式存在而是以二聚體或六聚體的多聚體形式存在，為此我們參考各個文獻設計了一套實驗，以驗證茲卡病毒NS1蛋白多聚體存在的情形。

Zika virus belongs to the genus Flavivirus of the Flaviviridae family. It was isolated from Uganda in Africa in 1947. In recent years, Zika virus has spread a large-scale epidemic in Central and South America and Southeast Asia. At the same time, many cases of fetuses born to pregnant women infected with Zika virus have a case of microcephaly, which has received attention. In the past, many flavivirus NS1 proteins associated with viral replication. In recent years, it has been pointed out that in addition to viral replication, flavivirus NS1 protein is released into the extracellular to interfere immune system and assists the virus infection in the initial stage. In summary, the NS1 protein of Zika virus should assist the viral replication or interfere host immune system to increase viral infectivity. Therefore, we observe the replication of viral RNA and expression of NS1 protein firstly, and then we overexpress the NS1 protein to confirm the impact of Zika viral life cycle. In addition, there is a lot of literature showing that the NS1 protein secreted to the outside of the cell is not in the form of a haploid but in the form of a dimer or a hexamer. For this we have designed a set of experiments which was performed to verify the polymer of Zika virus NS1 protein.

指導教授推薦書
口試委員會審定書
致謝 .........................................................iii
中文摘要 ......................................................iv
Abstract .......................................................v
目錄 ...........................................................vi
圖目錄 ........................................................ix
表目錄 ........................................................x
第一章 簡介 ......................................... - 1 -
一、茲卡病毒簡介 ........................... - 1 -
1. 分類 ......................................... - 1 -
2. 病毒流行病史........................... - 1 -
3. 病毒傳播與感染症狀............... - 2 -
4. 基因體與病毒蛋白................... - 3 -
5. 黃病毒生活史........................... - 3 -
二、非結構蛋白NS1 介紹 .............. - 5 -
1. 黃病毒NS1 蛋白 ................... - 5 -
2. 茲卡病毒NS1 蛋白 ............... - 5 -
3. 黃病毒NS1 蛋白檢測 ........... - 6 -
第二章 研究動機 ................................. - 8 -
一、茲卡病毒的大規模傳播與衍伸疾病 - 8 -
二、茲卡病毒的NS1 蛋白 .............. - 8 -
第三章 材料與方法（ Materials and Methods ） - 9 -
一、細胞培養 （Preparation of cell line） - 9 -
1. 解凍細胞 ................................. - 9 -
2. 細胞繼代 ............................... - 10 -
3. 冷凍細胞 ............................... - 11 -
二、茲卡病毒製備 （Zika Virus amplification） - 12 -
三、病毒斑實驗（Plaque assay） - 12 -
四、病毒感染 （Virus infection） - 13 -
五、RNA 萃取（RNA extraction） - 14 -
六、反轉錄聚合酶連鎖反應 （RT-PCR） - 14 -
七、即時聚合酶連鎖反應（Real-time Polymerase Chain Reaction）… - 15 -
八、SDS 聚丙烯醯胺膠體分離法（sodium dodecyl sulfate polyacrylamide gel
electrophoresis, SDS-PAGE） ...... - 16 -
九、西方墨點法（Western Blot） - 17 -
十、瓊脂糖膠電泳（Agarose gel electrophoresis） - 18 -
十一、純化DNA（Gel isolation & PCR clean up） - 19 -
1. Gel isolation： ..................... - 19 -
2. PCR clean up： ................... - 20 -
十二、Vector 與Insert DNA 接合（ligation） - 20 -
十三、質體轉型（Transformation） - 21 -
十四、質體DNA 製備（Plasmid DNA preparation） - 21 -
1. PlasPrep ............................... - 21 -
2. EndoFree .............................. - 22 -
十五. 質體轉染（Transfection） . - 23 -
十六. 分離細胞膜與細胞質的蛋白質 - 24 -
第四章 結果（Results） ................... - 25 -
1. 茲卡病毒感染BHK-21 細胞株的細胞存活率與病毒RNA 表現…… - 25 -
2. 茲卡病毒RNA 分密緻細胞外 . - 25 -
3. 茲卡病毒感染BHK-21 細胞株病毒蛋白的表現 - 26 -
4. 過量表現茲卡病毒NS1 蛋白 .. - 27 -
5. 茲卡病毒NS1 蛋白的多聚體 .. - 29 -
6. 茲卡病毒 NS1 蛋白常態性地分布在細胞的膜上 - 30 -
第五章 討論 ....................................... - 31 -
1. 建立茲卡病毒RNA 的生長曲線與感染後不同時間點的病毒蛋白- 31 -
2. 茲卡病毒NS1 蛋白會主動地釋放至細胞外 - 32 -
3. 過量表現茲卡病毒NS1 蛋白，試著觀察對茲卡病毒的生活史是否有影
響 ............................................. - 33 -
4. 茲卡病毒NS1 蛋白六聚體與細胞內的NS1 蛋白分布位置 - 34 -
第六章 圖表 ....................................... - 35 -
第七章 參考文獻 ............................... - 54 -


圖目錄
圖一 茲卡病毒細胞存活率與qPCR 換算用的standard curve - 36 -
圖二 茲卡病毒在BHK-21 細胞株內Growth Curve 和釋放至細胞外的病毒
RNA (病毒顆粒) ............................. - 38 -
圖五 在BHK-21 細胞株內過量表現茲卡病毒NS1 蛋白 - 43 -
圖六 茲卡病毒感染過量表現茲卡病毒NS1 蛋白的BHK-21 細胞株 - 47 -
圖七 茲卡病毒NS1 蛋白多聚體的偵測與分析 - 49 -
圖八 確認茲卡病毒NS1 蛋白常態分布的位置 - 51 -

表目錄
表一 實驗用引子 ........................... - 52 -
表二 pCMV - 3 Tag - 8 ................. - 53 -

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