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研究生:熊慧叡
研究生(外文):Hui-Jui Shiung
論文名稱:白點症病毒結構性蛋白質VP11(ORF394)之特性分析
論文名稱(外文):Characterization of White Spot Syndrome Virus (WSSV) Structural Protein VP11 (ORF394)
指導教授:張雲祥張雲祥引用關係
指導教授(外文):Yun-Shiang Chang
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:80
中文關鍵詞:白點症病毒VP11結構性蛋白質蛋白質交互作用自體激活
外文關鍵詞:white spot syndrome virus (WSSV)VP11structural proteinprotein interactionautoactivation
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白點症病毒(White spot syndrome virus, WSSV)是一個具有套膜的病毒,白點症病毒顆粒由三個結構層:外套膜、中間層和核鞘蛋白,圍繞它的核心DNA所構成。本研究針對白點症病毒結構性蛋白質VP11 (WSSV-T1 ORF394, GenBank accession no. AF440570)從事其特性分析。純化病毒顆粒以不同鹽濃度之Triton X-100溶液剝離其結構後以西方墨點法(Western blot)以及免疫電顯分析,得知VP11為一外套膜蛋白;膜拓璞學預測分析中顯示,VP11蛋白質在N端有高疏水性的穿膜區,而其C端則完全暴露於膜外,而以Sf9昆蟲細胞表現重組VP11並利用免疫螢光分析也得到與上述預測相同的結果,然而直接於病毒顆粒作用則得到相反的結果。本實驗室先前的研究解析出白點症病毒幾個主要結構性蛋白質間的交互作用,因此本研究也將VP11與這些已知關係的結構性蛋白質兩兩進行免疫沉澱(Co-immunoprecipitation)分析,結果發現VP11與外套膜蛋白質VP51A (WSSV-T1 ORF294)以及與其自身皆有交互作用。在酵母菌雙雜交分析結果證實VP11具有自體激活之特性,推測VP11可能為一轉錄因子,因此VP11可能在白點症病毒的生活週期,如病毒顆粒組裝和調控病毒基因表現扮演重要的角色。
White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that are surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. This study characterized a WSSV structural protein, VP11 (WSSV-T1 ORF394, GenBank accession no. AF440570). Immuno electromicroscopic analysis and Western blot hybridization of the intact viral particles and the separated viral components, the results showed that VP11 is an envelope protein. Membrane topology predition demonstrated that VP11 is a type transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal, and a C-terminal exposed on the virion surface. Immunofluorescence assay performed on VP11 transfected Sf9 cells demonstrated a similar result. However, trypsin digestion analysis of virions gave a controversial conclusion. Binary Co-immunopresipitation assays performed between VP11 and other a major WSSV structural proteins, respectively, VP11 was found to interact with both VP51A and itself. Yeast two-hybrid assay revealed that VP11 possessed autoactivation ability. We hypothesized that VP11 might act as a transcriptional factor. VP11 thus might play an important role in the replication of WSSV, not only in viral particle assembly but also in regulating at viral gene expression.
封面內頁
簽名頁
中文摘要 iii
英文摘要 iv
誌謝 v
目錄 vi
圖目錄 ix
表目錄 xi

1. 前言 1
1.1. 白點症病毒 1
1.2. 白點症病毒結構性蛋白質重要性 3
1.3. 蛋白質交互作用重要性 3
1.4. 白點症病毒已知有交互作用之結構性蛋白質 4
1.5. 酵母菌雙雜交系統之自體激活特性 4
1.6. 研究目的 5
2. 材料方法 6
2.1. 實驗流程 6
2.2. 基因表現分析 8
2.2.1. RNA萃取 8
2.2.2. 反轉錄聚合酶鏈鎖反應(reverse transcription
polymerase chain reaction, RT-PCR) 8
2.2.3. 基因時序表現分析 9
2.3. VP11於病毒顆粒定位之分析 10
2.3.1. 白點症病毒純化 10
2.3.2. VP11抗體與白點症病毒顆粒之西方墨點法分析 11
2.3.3. 以不同鹽濃度與Triton X-100剝離分析VP11 於
白點症病毒顆粒之定位 12
2.3.4. 以免疫電顯分析VP11於白點症病毒顆粒之定位 13
2.4. VP11模拓璞學(membrane topology)分析 14
2.4.1. VP11蛋白質親水性、疏水性分析 14
2.4.2. VP11穿膜區分析 14
2.4.3. 重組VP11(recombinant VP11, rVP11)於秋行軍蟲
細胞內(Spodoptera frugiperda, Sf9)之表現定位分
析 14
2.4.3.1. 質體DNA構築 14
2.4.3.2. 免疫螢光分析 15
2.4.4. VP11在白點症病毒顆粒上之膜拓璞學 16
2.5. VP11與其他白點症病毒結構性蛋白質交互作用分析 17
2.5.1. 免疫共沉澱分析 17
2.5.1.1. 質體DNA構築 17
2.5.1.2. 蛋白質表現 18
2.5.1.3. 共免疫沉澱分析 18
2.5.2. 遠方墨點法分析 20
2.5.2.1. 質體DNA構築 20
2.5.2.2. 蛋白質誘導表現 20
2.5.2.3. 蛋白質表現時序分析 21
2.5.2.4. pET28b-VP11重組蛋白之水溶性測試 21
2.5.2.5. 蛋白質小量純化分析 22
2.5.2.6. 蛋白質復性 22
2.6. VP11為轉錄活化因子分析 23
2.6.1. VP11蛋白質於酵母菌雙雜交系統中自體激活測試 23
2.6.1.1. 載體構築 23
2.6.1.2. 酵母菌轉染 23
2.6.1.3. 自體激活測試 24
3. 結果與討論 25
3.1. 基因分析 25
3.2. 基因表現時序分析 25
3.3. VP11於病毒顆粒定位之分析 26
3.4. VP11膜拓璞學分析 27
3.5. VP11 與其他白點症病毒結構性蛋白質交互作用 28
3.5.1. 共免疫沉澱分析VP11蛋白質與其它結構性蛋白質
交互作用 28
3.5.2. 遠方墨點法分析 29
3.6. VP11轉錄活化因子分析 30
4. 結論 32
參考文獻 70
附錄 75


圖目錄

圖1. 白點症病毒vp11基因mRNA轉錄表現時序分析 33
圖2. 西方墨點法分析VP11在白點症病毒顆粒上定位 34
圖3. 白點症病毒免疫電顯分析 35
圖4. 白點症病毒VP11蛋白親水性、疏水性預測 36
圖5. VP11穿膜區預測(TMHMM) 37
圖6. Sf9分析重組VP11之膜拓樸學 38
圖7. VP11膜拓璞學示意圖 39
圖8. VP11蛋白質之病毒拓璞學分析 40
圖9. 西方墨點法分析細胞中表現融合V5和FLAG tag之
VP11蛋白質 41
圖10. 白點症病毒結構性蛋白質VP11/VP19交互作用 42
圖11. 白點症病毒結構性蛋白質VP11/VP24交互作用 43
圖12. 白點症病毒結構性蛋白質VP11/VP26交互作用 44
圖13. 白點症病毒結構性蛋白質VP11/VP28交互作用 45
圖14. 白點症病毒結構性蛋白質VP11/VP32交互作用 46
圖15. 白點症病毒結構性蛋白質VP11/VP37交互作用 47
圖16. 白點症病毒結構性蛋白質VP11/VP38A交互作用 48
圖17. 白點症病毒結構性蛋白質VP11/VP11交互作用 49
圖18. 白點症病毒結構性蛋白質VP11/VP51A交互作用 50
圖19. 白點症病毒結構性蛋白質VP11/VP51A交互作用之再
確認 51
圖20. 白點症病毒結構性蛋白質VP11/VP51A_N、M、C端
交互作用 52
圖21. 重組VP1181-433蛋白質表現測試 55
圖22. 重組VP1181-433蛋白質表現時序分析 56
圖23. 重組VP1181-433蛋白質水溶性測試 57
圖24. 將1.5% sarcosine作用後重組VP1181-433蛋白質小量離
心純化測試 58
圖25. 將8 M urea作用後重組VP1181-433蛋白質小量離心純化
測試 59
圖26. 重組VP1181-335蛋白質表現測試 60
圖27. 重組VP1181-335蛋白質表現時序分析 61
圖28. 重組VP1181-335蛋白質水溶性測試 62
圖29. 重組VP1181-335蛋白小量離心純化測試 63
圖30. 白點症病毒結構蛋白VP11酵母菌雙雜交系統自體激活 64


表目錄

表1. 基因時序表現分析之特定核酸引子 65
表2. 構築於細胞中表現蛋白質載體引子 66
表3. 構築蛋白質表現載體引子 67
表4. 構築於酵母菌中表現蛋白質載體引子 68
表5. 共免疫沉澱分析蛋白質交互作用統整表 69
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