臺灣博碩士論文加值系統

蚊子是許多傳染病的主要病媒，舉凡瘧疾、絲蟲病、日本腦炎、登革熱、屈躬病，乃至近年爆發的茲卡病毒感染症 (Zika virus infection)，皆是經由不同種類的病媒蚊感染人類。目前茲卡病毒的防治多利用噴灑殺蟲劑控制病媒蚊數量作為主要降低病毒傳播的方式，並無有效之疫苗及藥物可供預防及治療。而針對突變率較高之RNA病毒設計藥物也易有抗藥性的產生，因此積極開發新穎替代性策略是首要之務。過去的文獻指出，茲卡病毒的非結構性蛋白 (nonstructural proteins, NSs) 會參與病毒自身的複製與組裝，並透過活化螺旋酶 (helicase) 與蛋白酶 (protease) 進行病毒蛋白的修飾。而在哺乳動物及昆蟲的模式研究中，宿主細胞的熱休克蛋白90 (Heat shock protein 90, Hsp90) 不僅會與茲卡病毒NS3蛋白有交互作用，當Hsp90被降解，也會使病毒複製能力下降。因此本研究將利用埃及斑蚊為研究模式，探討病媒蚊體內Hsp90與茲卡病毒NS3作為抑制病毒複製標的蛋白的可能性。從初步的實驗結果中，我們發現Hsp90與NS3不僅有形成複合物的現象，且Hsp90的表現受到抑制時，也會影響茲卡病毒的感染率。此外我們利用病毒蛋白抑制劑 (Hydroxychloroquine) 抑制NS3被後會影響Hsp90蛋白的表現。因此，我們推測茲卡病毒可能透過NS3蛋白切割的方式，改變宿主體內Hsp90表現，藉此進行調控病毒複製與增殖的能力。我們預期透過抑制Hsp90能干擾NS3發揮正常功能，因此降低病毒數量及感染率。未來我們也會經由調控Hsp90的表現，更進一步去了解茲卡病毒與埃及斑蚊體內蛋白間調控的分子機制，期許能為研發抗病毒藥物提供一個可能的方向。

The mosquito is the main vector of several important arthropod-borne diseases, such as malaria, filariasis, Japanese encephalitis, dengue fever, chikungunya and Zika. These diseases are transmitted from human to human by different mosquito vectors. However, no effective vaccine and medication are available for Zika virus infection until now. Currently, vector control through insecticide treatment become the main approach to reduce the transmission of Zika virus. However, the fast modification on RNA viral genome might neutralize the effect of inhibition. Therefore, development of alternative strategies for mosquito-borne disease control is urgently required. Previous reports indicated that ZIKV nonstructural proteins (NSs) participated in the viral replication and protein assembly, whereas the viral protein processing was mediated by non-structure protein 3 (NS3) through helicase and protease activity. In addition, NS3 has been shown to interact with heat shock protein 90 (Hsp90) in mammalian model. When hsp90 be silenced in insect cell, the virus replication be suppressed. Our preliminary results showed that Hsp90 interacted with NS3, and viral infectivity was reduced by inhibition of Hsp90 through reverse genetic approach. We also showed that inhibition of NS3 by hydroxychloroquine resulted in the inhibition of Hsp90 expression. We hypothesize that NS3 might play important role of modulating Hsp90 function through its protease activity. In the future, we will further investigate the detail mechanism underlying the regulation between ZIKV protein and host heat shock proteins. Information gathered in this study will pave the way toward the drug development by using molecular engineering approaches.

致謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 viii
表目錄 ix
第一章 緒論 1
1.1. 病媒蚊傳播疾病 (Mosquito-borne disease) 1
1.1.1. 台灣的蚊媒疾病 (Mosquito-borne disease in Taiwan) 3
1.1.1.1. 瘧疾 (Malaria) 3
1.1.1.2. 登革熱 (Dengue fever) 3
1.2. 埃及斑蚊生活史 (The life cycle of Aedes aegypti) 4
1.3. 茲卡病毒感染症 (Zika virus infection) 5
1.3.1. 臨床症狀 5
1.3.2. 國際之流行病學 5
1.3.3. 茲卡病毒生活史 6
1.3.3.1. 宿主細胞細胞內之複製 6
1.3.3.2. 病媒蚊體內之複製 6
1.4. 非結構性蛋白 (Non-structural protein 3，NS3) 參與病毒蛋白之成熟 7
1.5. 熱休克蛋白 (Heat shock proteins) 8
1.5.1. 熱休克蛋白家族 (Heat shock protein family) 8
1.5.1.1. 熱休克蛋白90 (Heat shock protein 90) 8
1.5.1.2. 熱休克蛋白70 (Heat shock protein 70) 9
1.5.1.3. 熱休克轉錄因子 1 (Heat shock transcription factor 1, HSF1) 9
1.6. 實驗動機 10
第二章 實驗材料與方法 11
2.1. 實驗步驟與流程 11
2.1.1. 埃及斑蚊之飼養與繼代 11
2.1.2. 病毒培養 (Virus culture) 12
2.1.3. 埃及斑蚊之餵血 12
2.1.4. 雙股RNA (double-stranded RNA, dsRNA) 製備 12
2.1.4.1. 質體建構 (plasmid construction) 12
2.1.4.2. 雙股RNA合成 (dsRNA synthesis) 13
2.1.5. RNA萃取 (RNA extraction) 14
2.1.6. 反轉錄作用 (reverse transcription, RT) 14
2.1.7. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 15
2.1.8. 即時定量聚合酶連鎖反應 (real-time PCR, Quantitative PCR, q-PCR) 15
2.1.9. 埃及斑蚊胸部注射 (Injection) 15
2.1.9.1. 病毒注射 (Virus injection) 15
2.1.10. RNAi-mediated silencing 與 knock-down efficiency test 16
2.1.11. 西方點墨法 (Western blot analysis ) 16
2.1.12. Hsp90 抑制試驗 (Hsp90 inhibiton assay) 17
2.1.13. 細胞培養 (Cell culture) 17
2.1.14. 溶斑試驗 (Plaque assay) 18
2.1.15. 免疫螢光染色法 (Immunofluorescence assay, IFA) 18
2.1.15.1. 組織免疫螢光染色法 18
2.1.15.2. 細胞免疫螢光染色法 19
2.1.16. 免疫沉澱試驗 (Immunoprecipitation , IP) 20
2.1.17. NS3抑制試驗 (NS3 inhibiton assay) 20
2.1.18. 細胞轉染 (Transfection) 21
2.2. 實驗試劑之配置 22
第三章 結果 24
3.1. 確認埃及斑蚊與人類之Hsp90蛋白序列相似性 24
3.2. 埃及斑蚊吸血方式感染ZIKV後Hsp90之表現情形 24
3.3. RNAi抑制Hsp90之效率以及對ZIKV表現量之影響 25
3.4. 抑制Hsp90對於ZIKV之感染力影響 26
3.5. ZIKV與Hsp90於Midgut、Salivary gland中位置相關性 27
3.6. 探討ZIKV病毒蛋白與埃及斑蚊Hsp90蛋白間之交互作用 28
3.7. 抑制Hsp90對於ZIKV之NS3於Midgut、Salivary gland中表現 28
3.8. 以Hsp90 inhibitor抑制Hsp90對於ZIKV成熟過程中的影響 29
3.9. 以NS3 inhibitor抑制ZIKV病毒蛋白表現對於Hsp90之影響 29
3.10. 設計Hsp90 HA-tag 的Overexpression系統觀察Hsp90對NS3之影響 30
第四章 討論 31
4.1. 埃及斑蚊與人類Hsp90之相似性 31
4.2. 埃及斑蚊Hsp90之表現情形及表現位置 31
4.3. 埃及斑蚊Hsp90對ZIKV之影響 32
4.4. Hsp90在埃及斑蚊被ZIKV感染後參與病毒複製的過程 33
附圖 35
附表 65
參考文獻 69

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