日本腦炎病毒(Japanese encephalitis virus, JEV)是一種藉蚊子傳染的黃質屬病毒。日本腦炎病毒套膜蛋白(envelope protein)的第三區域（ED3），在病毒感染宿主細胞時，可能與宿主細胞表面蛋白質接受器結合有關。蛋白酶(NS2b3)其病毒第三非結構性蛋白除有蛋白酶作用功能，在登革熱病毒研究還被發現與細胞凋亡（apoptosis）有關。本論文的研究利用噬菌體表達技術來篩選能對這兩種日本腦炎病毒蛋白相互作用結合的神經細胞蛋白質，之後利用ELISA及共同免疫沈澱來做進一步確認蛋白質之間的交互作用。篩選結果與ED3蛋白質相互作用的噬菌體表現人腦細胞cDNA的蛋白質，rED3-46 蛋白質是與粒腺體核糖蛋白（mitochondrial ribosomal protein L34）有將近100%的相似性。另一個rED3-17 蛋白質具有 GPP的motif，此motif也存在於膠原蛋白(collagen)之中。之後，利用噬菌體、重組人腦細胞蛋白以來進行日本腦炎病毒蝕斑減降試驗。實驗結果發現確實會有抑制病毒感染細胞的現象。利用重組人腦細胞蛋白對bal b/c小鼠進行腹腔注射免疫，取得血清後對神經細胞進行免疫螢光染色，發現確實在施打重組人腦細胞蛋白rED3-17小鼠免疫血清，使神經細胞外部周圍會有均勻的螢光。而施打重組人腦細胞蛋白rED3-46則在細胞中刻意觀察到一點一點的顆粒狀螢光。NS2b3蛋白質相對應的噬菌體所表現的蛋白質經比對，含帶有與神經退化疾病蛋白質（DRPLA）相關的motif。此蛋白質可能為細胞凋亡途徑中被caspase所活化的蛋白質，導致細胞凋亡發生，因此也將神經細胞轉染蛋白酶質體後進行免疫染色及西方墨點法，發現確實有細胞凋亡途徑的蛋白質active-caspase-3的存在。鑑定是何種神經細胞蛋白質與日本腦炎病毒蛋白質的相互作用都將有助瞭解日本腦炎病毒感染細胞時的分子致病作用機制。

Abstract

Japanese encephalitis virus belonging to flavivirus infects human by mosquitos. The domain III of envelope protein is regarded as to integrate the surface protein receptors from the host cell .The non-structure protein(NS2b3-180a.a) not only modifies protein but also relate the cellular apotosis. This study intends to identify cellular factors interacted with Japanese encephalitis virus (JEV) using phage display technology ,and then use ELISA and co-immunoprecipitation to further confirm the function. The result of Biopanning one group of ED3 associated proteins is relation with the mitochrondrial ribosomal protein L34 with 100% identy, the other group contains the GPP motif reported in the collagen. Functional interaction of ED3 protein with cellular factors ED3-17 and ED3-46 was examined using ELISA ,co- immunoprecipitation and plaque assay reduction test. Inaddition , the immunofluorescence staining of neuron cells was tested using the rED3-17 and rED3-46 immunized sera .The Biopanning result also indicated that most NS2b3-associated proteins show a motif ,which was found in dentatorubral-pallidoluysian atrophy (DRPLA). Apoptosis of the Neuron cells transfected with NS2b3-pcDNA3.1 was observed.The findings supported the induction of apoptosis by NS2b3 protein. Our results will be useful for understanding the molecular pathogenesis of Japanese encephalitis .

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