雄埃及斑蚊副腺蛋白Yeh317之生化特性分析、腺體內分布、與雌蚊交尾後之傳輸追蹤及副腺MAG-cDNA Library 建構之系列研究

雄埃及斑蚊副腺蛋白質可於交尾過程中傳輸到雌蚊體內刺激尋找宿主及產卵行為， 此雄性副腺內有一31.7 kDa 蛋白亦具有促使雌蚊拒絕交尾及刺激產卵行為。本研究為了要建構雄埃及斑蚊副腺蛋白質的cDNA Library；從1,000隻雄埃及斑蚊第八腹節中摘取副腺，純化蛋白、抽取Total RNA、篩檢出messenger RNA、合成單股DNA、雙股DNA，經由限制酵素EcoR I及Xho I之修飾，接合入載體pBluescript SK(+) phagemid 內，再以Gigapack III Gold Packaging Extract 吞包，此一MAG-cDNA的效價約200,000 plaques，再取E. coli MRF’ strain任其感染用以擴大此MAG-cDNA library，此一MAG-cDNA library是以E. coli strain XL1-Blue舖盤維持及採用Yeh317多株抗體篩檢；輔以山羊抗兔子IgG抗體鹼性磷酸酵素呈色，從此MAG-cDNA library中篩檢Yeh317蛋白殖株的工作到目前為止持續進行著。 本研究部分中是以SDS-PAGE, Coomassie brilliant blue染色檢視及純化Yeh317蛋白，並大量收集Yeh317蛋白用以注射及追加注射澳洲白鼻兔使其產生Yeh317多株抗體，再以西方點墨法檢測其抗體效價。 同時取大量Yeh317蛋白進行氨基酸N-端序列分析，此氨基酸N-端序列與埃及斑蚊 3al cDNA的中腸消化蛋白酵素原氨基酸N-端序列有47%的相似性。在雄埃及斑蚊副腺蛋白質的Yeh317蛋白特性分析中證實Yeh317蛋白為一可溶性蛋白質，進而再度以FITC in situ localization方法探討Yeh317蛋白在雄埃及斑蚊副腺分布及在交尾過程中的傳輸現象，證實Yeh317蛋白可經由交尾過程中傳輸到雌蚊儲精囊的現象；稱此現象屬於Venereal transmission。 在Yeh317蛋白質的生化功能探討方面；發現具活性的Yeh317蛋白能消化同種的雄埃及斑蚊副腺蛋白質 (MAG protein)，且能將果蠅 (Drosophila melanogaster) 中腸的GST蛋白分解並將之切為3段。 具活性的Yeh317蛋白在室溫(27℃) 中8小時內能分別將同種的雄埃及斑蚊副腺蛋白質及果蠅中腸的GST蛋白消化分解成片段，本研究之假設認為來自雄埃及斑蚊副腺蛋白質的Yeh317蛋白在已受精之雌蚊生理改變現象，及授精後雄蚊對她的無效交尾現象具有重要的意義。 本研究的第三部分是根據以上的實驗結果進一步探討雄埃及斑蚊副腺蛋白質中的Yeh317蛋白在交尾過程中傳輸入雌蚊體內的標示追蹤，本實驗以Yeh317多株抗體結合FISH的方法進行實驗探討，結果證實Yeh317蛋白在雌、雄蚊交尾過程中被傳輸入雌蚊的儲精囊中；此受孕雌蚊所產出的卵及孵化自此卵的一齡幼蟲皆可偵測得到Yeh317-FITC的螢光反應，由以上現象； 本研究之假設認為來自雄埃及斑蚊副腺蛋白質中的Yeh317蛋白在交尾過程中傳輸入雌蚊儲精囊中、受精卵、一齡幼蟲腦部中的lateral retractor of flabellum, median phageal ganglion, deutrocerebrum 及 protocerebrum等， 稱此現象為異性蛋白質經卵傳輸。The male accessory gland (MAG) protein that modulates the host-seeking behavior and stimulating oviposition of female Aedes aegypti (L.) mosquitoes were found to be a peptide of 31.7 kDa. This peptide also prevented subsequent mating behavior and weakly stimulated oviposition. For constructing cDNA library of male accessory gland of Ae. aegypti, RNA was isolated from adult male 3 days after feeding on sucrose, using the guanidinium thiocyanate extraction. Double stranded cDNA was synthesized using total RNA from 1,000 males as the template and directly cloned into the pBluescript SK(+) phagemid and packaged using the Gigapack III Gold Packaging Extract. Approximately 200,000 plaques were obtained and used to infect E. coli strain MRF' in order to amplify the library. The library was plated using E. coli strain XL1-Blue and screened with the mosquito 31.7 kDa (Yeh317) polyclonal antibody, and goat anti-rabbit IgG conjugated with alkaline phosphatase. Screening of the cDNA library is still undertaking at present. The Yeh317 protein was purified and characterized from the mosquito, Ae. Aegypti, by SDS-PAGE and Coomassie brilliant blue staining. The specificity of the polyclonal antibodies raised in rabbits to the purified Yeh317 protein was verified by immunoblot analysis of crude male accessory gland extracts and the protein was separated by SDS-PAGE. The N-terminal amino acid sequence of the purified mosquito male accessory gland 31.7 kDa protein (15 amino acids) had 79% identity to the derived amino terminal sequence of the CG6041 gene product of Drosophila melanogaster and 47% identity to the derived amino terminal sequence of 3al cDNA of midgut of Ae. aegypti that codes for a putative trypsinogen. The protein is a soluble protein and detected only in the supernatant of tissue homogenate. For in situ localization of the protein, laser-scanning microscope (Confocal) was used to observe cross-sections of MAG. This protein was found as distributed in the whole accessory gland. Subsequent work labeled males with goat anti-rabbit conjugated FITC to the protein and, allowed these males to copulate with virgin females. Detection of fluorescence reaction in female spermathecae demonstrates that the protein transferred during copulation. The crude MAG protein plays like a protease ability to digest GST protein. MAG protein could be digested with Yeh317 protein, therefore, use biochemistry method of in vitro study to prove that the protein from male accessory gland of Ae. aegypti which could destroy another MAG protein of Ae. aegypti, that demonstrate the protein could play like a protease activity to digest denature MAG protein, denature Yeh317 protein and Drosophila GST protein. Under room temperature condition of 27℃, the protein could digest GST protein and denatured Yeh317 protein. It might infer that in nature condition the female mosquito receptivity to further mating and, removing or destroying sperm of previous mates. The MAG Yeh317 protein landmarks have been developed so that this protein is transferred during mating. It was also demonstrated that Yeh317 protein could transfer during venereal transmission into spermathecae of gravid mosquitoes and carry over to her offspring, which unambiguously identified and oriented by fluorescence in situ hybridization (FISH) and digital imaging microscopy. In this study, it is indicated that the Yeh317 protein could be carried over to female mosquito during copulation, and the protein was found in embryo and ovaries. The protein even could be found in head part of lateral retractor of flabellum, median phageal ganglion, deutrocerebrum and protocerebrum of the hatched first instar larva.Chapter 1 A Male Accessory Gland Protein (Yeh317) in Aedes aegypti (L.): Isolation, Aminoterminal Sequence Determination and Localization. Chapter 2 The Function of Yeh317 protein of Male Accessory Glands in the Mosquito, Aedes aegypti (L.) Chapter 3 Fluorescence In Situ Hybridization of Aedes aegypti (L.) Male Accessory Gland Yeh317 Protein for Monitoring during Copulation