摘要
豬流行性下痢 ( Porcine epidemic diarrhea, PED )，是由豬流行性下痢病毒 ( Porcine epidemic diarrhea virus, PEDV ) 感染所導致，於全年齡豬隻皆會感染，感染後會在豬隻身上引起下痢、脫水、嘔吐及食慾不振等症狀。PED病例首次於西元1971年於英格蘭被注意，病毒則在西元1978年於比利時被分離出來。此病毒屬於冠狀病毒科 ( family Coronaviridae )的甲型冠狀病毒屬 ( genus Alphacoronavirus )，屬於單股RNA病毒，基因體長度約為28kb，能夠轉錄出四個結構蛋白，分別為大小約180-260kDa的棘突蛋白 ( Spike protein, S )、27-32kDa的膜蛋白 ( Membrane protein, M )、7kDa的封套蛋白 ( Envelope protein, E )、及55-58kDa的核蛋白 ( Nucleocapsid protein,N )。而其中S蛋白主要與受器結合並帶領病毒進入細胞，而且能夠於宿主體內誘發中和性抗體，也證實S蛋白上具有中和性抗原決定位 ( neutralizating epitopes )。另一方面，短芽孢菌 ( Brevibacillus choshinensis )目前常用來進行蛋白質的表現，此表線系統為革蘭氏陽性菌表現系統，優點在於其質體上帶有訊號胜肽 ( signal peptide )，能直接將目標蛋白分泌到培養液中，使我們在生產蛋白質時，可以不用進行破菌而得到產物。另外，此系統具有雙硫鍵的修飾，能使蛋白質產生摺疊，而更佳符合目標蛋白質原先的天然構形及可溶性。我們將以此陽性菌表現系統來生產PEDV的S蛋白作為次單位疫苗，並透過十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 ( SDS-PAGE )和西方墨點法 ( Western Blot )確認蛋白是可以以此表現系統表現的。搭配水包油包水佐劑 ( Water-in-oil-in-water, W/O/W )免疫於小鼠，接著進行酵素免疫分析 ( ELISA )及中和試驗 ( NT )，以評估此次單位疫苗的免疫能力，期望能對於PEDV具有保護性。
並列摘要
Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV). It would infect all age of swines, and be characterized by severe diarrhea, vomiting, and dehydration. PED was first reported in England in 1971 and the virus was first identified in Belgium. The virus belongs to the genus Alphacoronavirus, family Coronaviridae. It`s genome is single-stranded, positive-sense RNA which is contain 28 kb in length. The genome can translate four structure proteins, i.e. spike protein (S, 180-220kDa), membrane protein (M, 27-32kDa), envelope protein (E, 7kDa), and nucleocapsid protein (N, 55-58kDa). The spike protein plays the important role in receptor binding activity and elicts neutralizating antibody. And there are some studies prove evidences that spike protein contain neutralizating epitopes. In addition, Brevibacillus choshinensis expression system was use to produce recombinant protein in recent year. It is the gram-postive bacteria. Because the expression vector contains signal peptide, it can produce the recombinant protein in secreted form, and then we can harvest the protein without lysis bacteria and minimize lipopolysaccharide (LPS). B. choshinensis expression system has the ability to form a disulfide bond, which led the protein`s conformation close to native form. In this study, we used B. choshinensis expression system to produce PEDV spike protein to develop subunit vaccine, and through the result of SDS and western blot the protein could be produced by gram-positive bacterial expression system . We immunize the mice and harvest the serum to dectet the immune response by ELISA and neutralizating test.
參考文獻
延伸閱讀
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