- 學位論文
以基因轉殖蚊子防止登革病毒傳播
Prevention of Dengue Virus Transmission with Transgenic Mosquitoes
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摘要
登革病毒是一種正股RNA病毒，屬於黃病毒科。每年約有3.9億人感染登革病毒，其中1億人出現症狀，如發燒、肌肉疼痛、骨痛、出血性休克，甚至死亡。埃及斑蚊是主要的登革病毒傳播媒介。我們計劃製造攜帶登革病毒驅動致死基因的基因轉殖蚊子來阻止登革病毒的傳播。之前，我們的實驗室設計了一種報告系統，利用病毒蛋白酶-非結構蛋白 (NS) 3 介導在內質網 (ER) 錨定蛋白-NS4B和胞質蛋白-NS5 之間的肽鍵切割。重組蛋白包括NS4B和NS5的前10個氨基酸，被連接到含有核定位信號（NLS）的轉活化因子 (Gal4) 上，該因子結合上游啟動序列 (UAS)-熱休克蛋白70 (Hsp70) 啟動子並驅動eGFP的表達。同時，系統中還包括由埃及斑蚊眼睛特異性啟動子（3xP3）驅動的選擇標誌mCherry。我們在該設計中發現了兩個問題：1）Hsp70啟動子具有滲漏活性，將eGFP換成細胞凋亡致死基因後，即使Gal4不與UAS結合，Hsp70啟動子也可以驅動凋亡致死基因的表達，這可能會危及基因轉殖蚊子的適應性。2）3xP3啟動子驅動的mCherry使得篩選出基因轉殖蚊子變得困難。因此，我們將Hsp70啟動子改為已知能減少滲漏活性的果蠅合成核心啟動子（DSCP）。螢光顯微鏡和免疫印跡顯示，在沒有登革病毒感染的情況下，轉染DSCP啟動子的埃及斑蚊CCL125細胞中，eGFP的滲漏表達減少；為了促進基因轉殖蚊子的篩選，我們將3xP3啟動子替換為多聚泛素啟動子 (PUb promoter)。螢光顯微鏡和免疫印跡顯示，轉染多聚泛素啟動子的CCL125細胞中，mCherry信號強度大幅增加。多聚泛素啟動子登革熱病毒報告盒透過piggyBac轉座子系統被導入埃及斑蚊基因組中。胸腔注射使F2基因轉殖蚊子感染登革病毒而產生Hsp70啟動子驅動的eGFP信號。隨後進行的PCR和凝膠電泳確認了其中一個F2基因轉殖蚊子品系的基因組中插入了登革病毒報告盒。我們將登革病毒報告盒中的eGFP置換成靶向抑制凋亡蛋白1 （IAP1） 的microRNA-2 (miIAP1-2) 並將Hsp70啟動子改為低滲漏活性的DSCP啟動子，PCR和凝膠電泳，與隨後進行的Sanger定序，確認了F1基因轉殖蚊子的基因組中插入了DSCP 啟動子登革病毒驅動 miIAP1-2 表達盒。因此我們成功創建帶有DSCP 啟動子登革病毒驅動 miIAP1-2 表達盒之基因改造蚊子，預期登革病毒誘導的IAP1 knockdown會殺死這些基因改造蚊子。
並列摘要
Dengue virus is a positive-sense RNA virus belonging to the Flaviviridae family. Each year, approximately 390 million people are infected with dengue virus, with around 100 million experiencing symptoms such as fever, muscle pain, bone pain, hemorrhagic shock, and even death. Aedes aegypti is the primary vector for dengue virus transmission. We propose creating transgenic mosquitoes carrying a dengue virus-driven lethal gene to prevent the transmission of dengue virus. Previously, our laboratory designed a reporter system utilizing the viral protease nonstructural protein (NS) 3 to mediate peptide bond cleavage between the endoplasmic reticulum (ER)-anchored NS4B and the cytoplasmic NS5. The recombinant protein includes the first 10 amino acids of NS4B and NS5, fused to a Gal4 transactivator with a nuclear localization signal (NLS). This factor binds to the upstream activating sequence (UAS)- heat shock protein 70 (Hsp70) promoter to drive the expression of eGFP. The system also includes a selection marker, mCherry, driven by the Aedes aegypti eye-specific promoter (3xP3). We identified two issues with this design: 1) The Hsp70 promoter exhibits leaky activity, which, after replacing eGFP with an apoptotic lethal gene, can drive the expression of the lethal gene even if Gal4 does not bind to UAS. This could compromise the fitness of the transgenic mosquitoes. 2) The 3xP3 promoter-driven mCherry makes it challenging to select transgenic mosquitoes. To address these issues, we replaced the Hsp70 promoter with the Drosophila synthetic core promoter (DSCP), known to reduce leaky activity. Fluorescence microscopy and immunoblotting showed reduced leaky expression of eGFP in Aedes aegypti CCL125 cells transfected with the DSCP promoter in the absence of dengue virus infection. To facilitate the selection of transgenic mosquitoes, we replaced the 3xP3 promoter with the polyubiquitin promoter (PUb promoter). Fluorescence microscopy and immunoblotting revealed a significant increase in mCherry signal intensity in CCL125 cells transfected with the PUb promoter. The PUb promoter-based dengue virus reporter cassette was integrated into the Aedes aegypti genome using the piggyBac transposon system. Thoracic injection of dengue virus into F2 transgenic mosquitoes resulted in Hsp70 promoter-driven eGFP signals. Subsequent PCR and gel electrophoresis confirmed the integration of the dengue virus reporter cassette in one F2 transgenic mosquito line. We replaced eGFP in the dengue virus reporter cassette with microRNA-2 targeting the inhibitor of apoptosis 1 (miIAP1-2) and changed the Hsp70 promoter to the low-leakage DSCP promoter. PCR, gel electrophoresis, and Sanger sequencing confirmed the insertion of the DSCP promoter-based dengue virus miIAP1-2 expression cassette in the F1 transgenic mosquitoes. Thus, we successfully created transgenic mosquitoes carrying the DSCP promoter-based dengue virus miIAP1-2 expression cassette, with the expectation that dengue virus-induced IAP1 knockdown will result in the death of these transgenic mosquitoes.
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