臺灣博碩士論文加值系統

登革熱病毒屬黃熱病毒科(Flaviviridae)黃熱病毒屬(Flavivirus)。它會造成登革熱(dengue fever)、登革出血熱(dengue hemorrhagic fever)及登革休克症候群(dengue shock syndrome)等症狀。登革熱病毒產生蛋白質的過程中，會先產生單一個蛋白質轉譯區，再切割成10 個獨立的蛋白質。包含3 個結構性蛋白(structural proteins) 以及7 個非結構性蛋白(non-structuralproteins)，其中外膜蛋白(envelope protein)便屬於結構性蛋白，位於病毒雙層膜的外側，是一種穿膜的蛋白質，被認為可和受體結合，與感染宿主細胞有關。NS1 與NS2A、NS2B、NS4A、NS4B 是較小的非結構性蛋白，具有疏水的特性，皆是與膜有關的蛋白質。已知NS2B 會參與蛋白切割酶切割反應，而NS1、NS2A、NS4A、NS4B 則與病毒的複製有關。為研究蛋白質的功能，首先必須建構質體並於大腸桿菌系統中表現蛋白質，本研究將針對NS1、NS2A、NS2B、NS4A、NS4B 與E 蛋白等蛋白質做探討，由於這些蛋白質皆具有疏水的特性，因此選用大腸桿菌C41（DE3）及C43（DE3）菌株以利其表現。將基因構築於修飾過pET-30(+)表現載體：pET5T-HAHis上， 分別於基因末端接上融合蛋白HA-His(influenza Hemagglutinin-Hexahistidine protein)以利抗體偵測。將此建構質體送入大腸桿菌C41（DE3）或C43（DE3）菌株後，皆可採用西方點墨法(Western blot)偵測其表現，但以Comassie blue 染色則無法偵測到。

Dengue virus is a member of family Flaviviridae, genus of Flavivirus. Dengue viruses cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue virus encodes 10 proteins in a single open reading frame, including 3 structural proteins and 7 non-structural proteins. Envelope protein is one of the structural proteins locates outside of the virus particle, which can bind to receptors on host cells and causes infection. NS2A, 2B, 4A and 4B are small non-structural proteins which are membrane-associated proteins exhibiting hydrophobic profiles. NS2B has been suggested to involve in protease activity. NS1, NS2A, NS4A and NS4B have been suggested to involve in virus replication. Individual expression plasmids were constructed and expressed in E.coli C41（DE3）and C43（DE3） strains due to their hydrophobic profiles. These genes were cloned individually into a modified pET-30(+) vector, pET5T-HAHis. Their proteins were expressed as HA-His (influenza Hemagglutinin- Hexahistidine protein) fusion proteins for antibodies detection. Recombinant proteins fused with HA-His tag could be detected by Western blotting analysis, but not by Comassie blue staining.

目錄
中文摘要 I
Abstract II
致謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
附錄目錄 X
縮寫（Abbreviation） XI
壹、緒 論 1
1.1 登革熱病毒 1
1.2 登革熱病毒的基因結構 2
1.3 登革熱病毒基因轉譯 2
1.4 外膜蛋白E之特性 3
1.5 非結構蛋白NS1之特性 4
1.6 非結構蛋白NS2A的特性 5
1.7 非結構蛋白NS2B的特性 6
1.8 非結構蛋白NS4A的特性 6
1.9 非結構蛋白NS4B的特性 7
1.10 表現登革熱蛋白質的系統-大腸桿菌C41（DE3）與C43（DE3）菌株 7
貳、材料與方法 9
2.1 實驗材料 9
2.1.1 菌株（Bacteria Strains） 9
2.1.2 病毒（Viruses） 9
2.1.3 質體（Plasmids） 9
2.1.4 引子（Primers） 11
2.1.4.1 針對建構質體所設計的引子 11
2.1.4.2 針對Site-Directed Mutagenesis所設計的引子 13
2.1.5 藥品試劑 14
2.1.5.1 藥品 14
2.1.5.2 抗體 15
2.1.5.3 Kit 16
2.1.6 溶劑及緩衝液之配方 16
2.1.7 主要儀器 17
2.2 實驗方法 18
2.2.1 大腸桿菌勝任細胞（Competent cell）的製備 18
2.2.2 大腸桿菌轉形作用（transformation） 18
2.2.3 建構質體DNA 19
2.2.4 小量質體DNA萃取 19
2.2.5 限制酵素反應 20
2.2.6 萃取洋菜膠內之DNA片段 20
2.2.7 重組蛋白在大腸桿菌之誘導表現 20
2.2.8 大腸桿菌蛋白質之萃取 21
2.2.9 以SDS-PAGE分析並以Coomassie blue staining及西方轉漬分析(Western blot analysis)偵測蛋白質表現 21
2.2.9.1 SDS-PAGE電泳 21
2.2.9.2 Coomassie blue staining 21
2.2.9.3西方轉漬分析(Western blot analysis) 22
參、結果 23
3.1 pET△5T-HAHis質體之建構 23
3.2 pET△5T-D2E-HAHis, pET△5T-D2NS1-HAHis, pET△5T-D2NS1(d) -HAHis pET-30a-D2NS1-HAHis質體之建構 23
3.3 pET△5T-D32A-HAHis, pET△5T-D32B-HAHis, pET△5T-D34A -HAHis, pET△5T-D34B-HAHis, pET△5T-D3E-HAHis質體之建構 24
3.4利用限制酵素以確認所建構之質體 24
3.5 序列結果比對 26
3.6 登革熱病毒非結構蛋白以及外膜蛋白（E protein）在E.coli C41（DE3）和C43（DE3）中的表現 27
3.6.1登革熱病毒非結構蛋白NS2與NS4的表現 27
3.6.1.1 pET△5T-D2NS2A-HAHis, pET△5T-D3NS2A-HAHis蛋白質表現結果 27
3.6.1.2 pET△5T-D2NS2B-HAHis, pET△5T-D3NS2B-HAHis蛋白質表現結果 28
3.6.1.3 pET△5T-D2NS4A-HAHis, pET△5T-D3NS4A-HAHis蛋白質表現結果 29
3.6.1.4 pET△5T-D2NS4B-HAHis, pET△5T-D3NS4B-HAHis蛋白質表現結果 30
3.6.2登革熱病毒外膜蛋白的表現 31
3.6.2.1 pET△5T-D2E-HAHis, pET△5T-D3E-HAHis蛋白質表現結果 31
3.6.3登革熱病毒非結構蛋白NS1的表現 32
3.6.3.1 pET△5T-D2NS1-HAHis蛋白質表現結果 32
3.6.3.2 pET△5T-D2NS1(d)-HAHis蛋白質表現結果 33
3.6.3.3 pET30a-D2NS1-HAHis蛋白質表現結果 33
肆、討論 35
4.1 質體建構與序列分析 35
4.2表現登革熱病毒非結構蛋白NS2與NS4與外膜蛋白在Ecoli C41（DE3）和C43（DE3）表現系統 35
4.3表現登革熱病毒外膜蛋白在E.coli C41（DE3）和C43（DE3）表現系統 37
4.4表現登革熱病毒非結構蛋白NS1在E.coli C41（DE3）和C43（DE3）表現系統 37
伍、結 論 40
陸、參考文獻 41

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