陳維鈞吳文哲臺灣大學：黃旌集Huang, Chin-GiChin-GiHuang2007-11-262018-06-292007-11-262018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/55075簇蟲(Gregarine)是蚊蟲體內常見的寄生性原生動物，在分類上屬於原生物界(Protista)、頂複合器門(Apicomplexa)、簇蟲綱(Gregarinea)、真簇蟲目(Eugregarinida)、衣孢蟲科(Lecudinidae)、囊簇蟲屬(Ascogregarina)。其生活史完全在昆蟲體內完成，並與宿主同步發育。以寄生於白線斑蚊(Aedes albopictus)的臺灣簇蟲(A. taiwanensis)為例：卵囊體(oocysts)被蚊幼蟲食入後，在腸道中會釋出孢子小體(sporozoite)，並侵入中腸的上皮細胞，發育為細胞內營養體(trophozoite)，隨後離開宿主細胞，並以上節(epimerite)附著於宿主的上皮細胞上，在宿主化蛹的第一天，大多數的營養體離開宿主中腸，遷移至馬氏管，發育成為配子體(gamete)，當配子體配對後即發育成為配子囊體(gametocyst)，而產生為數甚多檸檬狀的卵囊體。根據實驗結果顯示，簇蟲卵囊體在pH 11的鹼性環境下才會釋出孢子體，而蚊子中腸pH值主要受腸道細胞的V-ATPase調控。V-ATPase是屬於耗ATP的質子幫浦，通常出現在真核細胞的內膜上，形成酸性的組織如溶小體、胞小體及parasitophorous vacuoles等，甚至一些具有套膜的病毒在感染細胞時需依賴V-ATPase所形成的酸化環境。但在蚊子腸道中，V-ATPase則扮演著使腸道形成鹼性環境的角色。在本研究中，將探討V-ATPase對簇蟲感染的影響，同時，偵測V-ATPase在宿主表現位置的差異，實驗中利用部份的白線斑蚊V-ATPase subunit A之序列，選殖出完整的V-ATPase subunit A基因並以大腸桿菌表現系統表現V-ATPase subunit A蛋白質，此完整的V-ATPase subunit A序列共有2791個核酸，具有一完整的ORF (open reading frame)，長度為1842個核酸，此ORF在pET30a載體上表現出來的蛋白質為74 kDa，利用此蛋白質去生產抗V-ATPase subunit A的血清，並利用此血清去檢測V-ATPase在腸道的表現，發現中腸前後段的V-ATPase表現位置不同，而切片的結果顯示簇蟲只寄生於中腸後段的細胞。另一方面，用酸性囊泡染劑Acridine orange染色，發現被簇蟲寄生的腸道中，簇蟲會在胞內形成酸性的囊泡。綜合上述結果發現V-ATPase可能影響簇蟲的寄生，於是用V-ATPase的專一性抑制劑bafilomycin A1抑制蚊幼蟲的V-ATPase功能，發現可顯著抑制簇蟲的寄生。此結果代表簇蟲的寄生需依賴宿主功能的正常的V-ATPase。Gregarines (Apicomplexa, Eugregarinida, Lecudinidae) are common parasites of mosquitoes. The life cycle of Ascogregarina is completed in the insect with its development synchronized with the host. The mosquito Aedes albopictus acquires the A. taiwanensis from the ingestion of its oocysts in the breeding water. Within the digestive tract, the sporozoites emerge from the oocysts and enter the epithelial cells. After a short duration of intracellular development, the trophozoite attaches to the luminal side of the midgut cell by the epimerite. During the mosquito pupation, the gregarine migrates into the Malpighian tubules to begin the gametogenous phase. The gregarine sporozoites are released by ingested oocysts in the high pH environment. In mosquito larvae, V-ATPase plays an important role in regulating luminal alkalization of midgut. The V-ATPase is a family of ATP-dependent proton pumps and usually located on the endomembrane of eukaryotic cells. Acidification of intracellular compartments such as lysosomes, endosomes, and parasitophorous vacuoles medicated by V-ATPase are essential for the entry of many enveloped viruses and invasion into or escape from host cells. We had cloned and expressed the full-length V-ATPase subunit A protein from mRNAs extracted from Ae. albopictus larval tissues. This open reading frame (ORF) and 3’ uncoding sequences were 2971 base pairs in length. The rabbit anti V-ATPase subunit A antiserum was prepared and was used to detect V-ATPase in the midgut of mosquito larvae. Antibody-binding sites were observed to be located at the basal of anterior midgut and the apical of posterior midgut. Longitudinal section of infected larva showed gregarine located in the posterior midgut. In response to the invasion of A taiwanensis, a parasitophorous vacuole confining intracellular trophozoites was formed; within which acidification was demonstrated by using acridine orange staining. Mosquito treated with bafilomycin A1, a specific inhibitors of V-ATPase, showed a dramatic reduction in intensity of infection by gregarina. Therefore, V-ATPase may play a crucial role in determining the infection of gregarine.中文摘要………………………………………………………………… i 英文摘要………………………………………………………………iii 目錄………………………………………………………………………v 表次……………………………………………………………………ix 圖次………………………………………………………………………x 壹、緒言………………………………………………………………1 貳、往昔研究……………………………………………………………3 一、簇蟲簡介………………………………………………………4 二、簇蟲生活史.................................................................................4 三、簇蟲的宿主專一性......................................................................4 四、蛻皮激素對簇蟲發育的影響......................................................6 五、其他寄生蟲研究..........................................................................7 (一)瘧原蟲.....................................................................................7 (二)巴貝斯蟲.................................................................................8 (三)蟠尾絲蟲.................................................................................9 六、V-ATPase簡介...........................................................................9 七、蚊子的V-ATPase.......................................................................10 八、病毒與V-ATPase的關係……………………………………………11 九、寄生蟲與V-ATPase的關係.......................................................12 參、材料與方法......................................................................................14 一、實驗室蟲原................................................................................14 (一)蚊種.......................................................................................14 (二)簇蟲來源...............................................................................14 二、蚊蟲飼養法................................................................................14 三、卵囊體純化................................................................................14 四、蚊感染強度測試........................................................................15 五、卵囊體不同pH值處理後梯度離心回收率實驗......................15 六、卵囊體掃瞄式電子顯微鏡樣本處理........................................15 七、冷凍切片....................................................................................16 八、核醣核酸萃取............................................................................16 九、反轉錄反應................................................................................17 十、聚合酶鏈反應............................................................................17 十一、限制酶切................................................................................17 十二、接合作用................................................................................18 十三、轉型作用................................................................................18 十四、快速篩選................................................................................18 十五、利用聚合酶鏈反應確認菌落...............................................18 十六、蛋白質表現及表現時間測試................................................19 十七、表現的蛋白質可溶性測試....................................................19 十八、蛋白質大量表現、純化及濃縮.........................................20 十九、抗血清製備............................................................................21 二十、斑蚊腸道細胞全蛋白抽取及純化........................................21 二十一、SDS-PAGE...................................................................21 二十二、ECL西方墨漬法...............................................................22 二十三、免疫組織化學....................................................................23 二十四、V-ATPase抑制劑bafilomycin A1處理對簇蟲感染強度之影響......................................................................................23 二十五、Acridine Orange 染色......................................................23 二十六、親緣關係分析方法……………………………………………23 肆、結果.......................................................................………………...25 一、卵囊體純化...............................................................................25 二、蚊感染強度測試........................................................................25 三、卵囊體不同pH值處理後梯度離心回收率實驗......................25 四、高pH值處理對卵囊體的影響..................................................26 五、冷凍切片...................................................................................26 六、V-ATPase subunit A之cDNA序列..........................................26 七、V-ATPase subunit A表現及純化...............................................28 八、抗V-ATPase subunit A血清效價及偵測白線斑蚊腸道中的V-ATPase subunit A...................................................................28 九、V-ATPase subunit A在腸道的分佈.......................................29 十、V-ATPase抑制劑bafilomycin A1處理對簇蟲感染強度之影響.........................................................................................29 十一、感染簇蟲的白線斑蚊腸道以Acridine orange染色之結果................................................................................................29 伍、討論...................................................................................................31 陸、引用文獻............................................................................................37 柒、誌謝………………………………………………………………………46 表............................................................................................................47 圖............................................................................................................494950995 bytesapplication/pdfen-US白線斑蚊臺灣簇蟲V-ATPaseAedes albopictusAscogregarina taiwanensisthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55075/1/ntu-94-D88632002-1.pdf