Charaterization of white spot syndrome virus (WSSV)structural protein VP11(ORF394) 熊慧叡、張雲祥
E-mail: [email protected]
ABSTRACT
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.
Keywords : white spot syndrome virus (WSSV)、VP11、structural protein、protein interaction、autoactivation Table of Contents
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? 嚍磥狨 部]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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