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研究生:郭倫慈
研究生(外文):Lun-Tzu Kuo
論文名稱:探討登革病毒在埃及斑蚊體內成熟之分子調控機制
論文名稱(外文):Study the molecular regulation of dengue virus maturation in Aedes aegypti
指導教授:蕭信宏蕭信宏引用關係
指導教授(外文):Shin-Hong Shiao
口試日期:2017-07-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:79
中文關鍵詞:埃及斑蚊登革病毒FurinVg convertase (VgC)prM
外文關鍵詞:Aedes aegyptiDengue virusFurinVg convertase (VgC)prM
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蚊子是許多疾病的傳播媒介,例如:瘧疾、登革熱、屈躬病、西尼羅熱、黃熱病以及茲卡病毒等。當中,登革熱為現今非常重要的議題,根據世界衛生組織統計顯示,全世界每年大約有3.9億的案例,其中約有9000萬的人口具有臨床症狀,而在台灣2014至2015年之間,即有超過5萬件的感染案例。由於目前仍無有效的藥物及疫苗上市,因此,如何預防病媒蚊的叮咬以及控制病媒蚊的數量,為當前防治蚊媒傳染疾病最重要的研究課題。根據先前研究文獻指出,人體中的一個蛋白質轉化酶 (proprotein convertases, Furin)對於登革病毒成熟的過程中扮演非常重要的角色,經序列比對後,我們發現埃及斑蚊的Vitellogenin convertase (VgC)與Furin具有高度的同源性,而VgC於蚊子吸血後大量表現並參與卵巢發育。因此,本研究以埃及斑蚊為研究模式,探討VgC對於登革病毒成熟的影響。首先,分別給予蚊子正常血液與含有病毒的血液,結果顯示VgC的表現量皆會上升,因此我們推測登革病毒本身並不會影響VgC的表現,而是可能直接利用蚊子吸血後表現的VgC來幫助病毒的成熟。此外,免疫螢光染色法的分析結果顯示,VgC與登革病毒之位置非常接近或是重疊,表示登革病毒有機會與VgC接觸。接著,我們利用Furin inhibitor抑制VgC之活性,發現經Furin inhibitor處理的埃及斑蚊,Vg蛋白質表現量有顯著的下降,由此可確認Furin inhibitor的效用,再者,我們更進一步探討抑制VgC之後,對於登革病毒成熟過程之影響,結果顯示注射Furin inhibitor的組別,其登革病毒的precursor membrane (prM)蛋白質表現量有明顯的上升,表示不成熟的登革病毒顆粒比例相對增加。本研究證實VgC對於登革病毒在成熟過程中所扮演的重要角色,本結果將對於未來在病媒傳播病毒之研究上有重要的幫助。
Mosquitoes are one of the fatal animals in the world and they act as vectors to carry and spread disease to humans, including malaria, dengue fever, West Nile fever, chikungunya and Zika. Among these diseases, dengue fever is an emerging threat in the world. In Taiwan, over 50 thousand cases have been reported in 2014 and 2015. However, there are no effective medication and vaccine for dengue fever available. Thus, developing strategies to fight against mosquito-borne diseases is urgently needed. Previous studies indicated that human Furin, a proprotein convertases, plays an important role in dengue virus (DENV) maturation. We identified a Vitellogenin convertase (VgC) in the mosquito Aedes aegypti to be highly homologous to Furin. VgC has been described to be involved in the production of vitellogenin (Vg) during mosquito vitellogenesis. The aim of this study is to investigate the effect of VgC on DENV maturation. We showed that the VgC transcript was highly expressed after a normal and an infectious blood meal, suggesting that DENV may make use of blood meal-induced VgC for replication. In addition, VgC was found to co-localize with DENV after infection, suggesting that DENV had opportunity to use VgC. Next, we used furin inhibitor to study the effect of VgC in viral replication. To confirm the efficiency of furin inhibitor, we examine the expression of vitellogenin post furin inhibitor treatment. Furthermore, the expression of precursor membrane (prM) protein of DENV was increased after furin inhibitor treatment, indicating that VgC was responsible for DENV maturation. Our results provide new insights into the understanding of DENV and vector interaction which may help to devise a new strategy to eradicate DENV program.
誌謝 ii
中文摘要 iii
Abstract iv
目錄 v
圖目錄 ix
表目錄 x
第一章 緒論 1
1.1. 病媒蚊傳播疾病 (Mosquito-borne disease) 1
1.1.1. 瘧疾 (Malaria) 3
1.1.2. 茲卡病毒感染症 (Zika virus infection) 3
1.2. 登革熱 (Dengue fever) 4
1.2.1. 登革病毒 (Dengue virus,DENV) 4
1.2.2. 臨床症狀 4
1.2.3. 台灣地區之流行病學 5
1.3. 埃及斑蚊生活史 5
1.4. 登革病毒生活史 6
1.5. Furin 7
1.6. 埃及斑蚊之Vg convertase (VgC) 9
1.7. 實驗動機與假說 9
第二章 實驗材料與方法 11
2.1. 實驗步驟與流程 11
2.1.1. 埃及斑蚊飼養與繼代 11
2.1.2. 細胞培養 (Cell culture) 11
2.1.3. 病毒製備 12
2.1.4. 埃及斑蚊之餵血 (Blood feeding) 12
2.1.5. 雙股RNA (double-stranded RNA, dsRNA) 製備 12
2.1.6. RNA萃取 (RNA extraction) 14
2.1.7. 反轉錄作用 (Reverse transcription, RT) 14
2.1.8. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 15
2.1.9. 即時定量聚合酶連鎖反應 (Real-time PCR, Quantitative PCR) 15
2.1.10. 埃及斑蚊之胸腔注射 (Injection) 16
2.1.11. 西方點墨法 (Western blot) 16
2.1.12. 免疫螢光染色 (Immunoflorescent assay,IFA) 17
2.1.13. 埃及斑蚊唾液 (saliva)收取 18
2.1.14. 溶斑試驗 (Plaque assay) 18
2.1.15. 建立CRISPR-Cas9 VgC knock out 系統 19
2.1.16. pAc5.1-VgC質體建構 20
2.2. 實驗試劑製備 21
第三章 結果 24
3.1. 確認埃及斑蚊之VgC與人類之Furin序列相似性 24
3.2. 埃及斑蚊感染DENV2後VgC之表現情形 25
3.2.1. 以顯微注射方式感染後VgC於各組織之表現情形 25
3.2.2. 以吸血方式感染後VgC整體之表現情形 25
3.2.3. 以吸血方式感染後VgC於各組織之表現情形 26
3.3. VgC於埃及斑蚊Fat body與Ovary中之表現位置 26
3.4. DENV2與VgC於Fat body中表現位置之相關性 27
3.5. DENV2與VgC於Midgut、Salivary gland、Ovary中之位置相關性 27
3.6. DENV2、VgC與Golgi於Fat body與唾腺中之位置相關性 28
3.7. RNAi抑制VgC之效率以及對DENV2表現量之影響 29
3.8. 以Furin inhibitor抑制VgC確認其與Furin之相似性 29
3.9. 抑制VgC對於DENV2成熟過程中prM切割之影響 30
3.10. 抑制VgC後對於唾液中DENV2之感染力影響 31
3.11. 利用pAc5.1-VgC質體探討VgC對於DENV之影響 31
3.12. 以ATC10細胞探討抑制VgC對於DENV2之影響 32
3.13. 以CRISPR-Cas9建立埃及斑蚊VgC-knock out系統 33
第四章 討論 35
4.1. 埃及斑蚊VgC與人類Furin之相似性 35
4.2. 埃及斑蚊VgC之表現情形及表現位置 35
4.3. VgC與DENV2於各組織之表現關係 36
4.4. VgC對於DNEV2之影響 37
4.5. 埃及斑蚊中其他兩種類似VgC之protein:AAEL014523與AAEL010725 39
附圖 40
附表 68
附錄 71
參考文獻 74
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