第71型腸病毒好發於每年夏季期間，其易引起手足口病症，而近年來在台灣爆發過兩次的腸病毒大流行，並且在1998年與2000年造成了78名與34名孩童死亡。在全球的流行病學上有跡象顯示近幾年腸病毒多在亞洲與非洲區域造成流行，而目前在腸病毒之治療上並沒有可用之特效藥，只能採用支持性療法，故疫苗之研發為當務之急。第71型腸病毒為一不具外套膜之小RNA病毒，其外鞘蛋白由VP1、VP2、VP3與VP4所組成，而VP1為具有抗原決定性之外鞘蛋白，VP4為一連結角色將VP1、VP2與VP3組合形成一完整的病毒顆粒。在本篇試驗中，將一已分離之第71型腸病毒經定序比對其外鞘蛋白VP1至VP4胺基酸序列後，結果顯示其相似度與新加坡兩腸病毒株(AF316321與AF352027)最為相近，高達98.96%，且推測這些改變乃為第71型腸病毒之突變熱區(hot spot)所在區域。我們並將第71型腸病毒外鞘蛋白VP1以基因轉殖之技術產製出基因轉殖鼠，並藉由乳腺專一性的表現載體內含α-白蛋白啟動子與αS1酪蛋白訊息引導序列將VP1蛋白成功的表現與釋泌於鼠乳中；我們也建立起第71型腸病毒攻毒之動物模式，對4天大之仔鼠以腹腔注射進行攻毒試驗會與對照組仔鼠其體重產生明顯差異。血清中和試驗法也更進一步證實，基因轉殖鼠其仔鼠可因吸食帶有第71型腸病毒外鞘蛋白VP1之鼠乳而誘發其特異性之免疫反應，進而對第71型腸病毒產生具專一性之抗體。Enterovirus 71 (EV71) is the most common aetiological agent detected in cases of hand, foot and mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. It had been out- break twice in Taiwan which caused 78 and 34 infected patient deaths in 1998 and 2000, respectively. The clinical data have been shown that the significant increase in EV71 epidemic activity throughout the Asia-Pacific region recently. Because of the lack of an effective antiviral agent, the primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. The out-layer of EV71 viral particle is composed by four capsid proteins, VP1, VP2, VP3 and VP4. The VP1 has a major antigenic coat protein, while the VP4 acts as a linker protein anchoring VP1 to VP3 and forms a complete viral particle. In this study, we first cloned a new indentified EV71 virus and its amino acid sequence of capsid proteins were found highly homology with strains from Singapore (AF316321 and AF352027), then a mammary gland-specific expression transgenic animal system harboring EV71 capsid protein genes has been established to investigate the feasibility of EV71 capsid proteins production in transgenic milks. The results showed that the VP1 and proteins can be expressed and secreted into transgenic milk in lactating period via the control of alpha-lactalbumin promoter and alpha-casein leader sequences. And according to serum-neutralization assay, these littermates suckling the transgenic milk were gernerated the antibody specific to EV71. We were also establishiment the animal model for EV71 challenge assay.圖次………………………………………………………………………………3
表次………………………………………………………………………………5
壹、中文摘要……………………………………………………………………6
貳、緒言…………………………………………………………………………8
參、文獻探討……………………………………………………………………10
一、腸病毒之分類與基因結構……………………………………10
二、感染腸病毒所引起之臨床病症………………………………16
三、第71型腸病毒之重症感染與治療策略………………………23
四、基因轉殖動物模式之開發……………………………………32
五、基因轉殖動物之乳腺表現型載體系統………………………40
六，利用乳腺表現型基因轉殖動物進行第71型腸病毒外鞘蛋
白之表現及其於口服性疫苗開發之評估……………………………………43
肆、材料與方法………………………………………………………………45
伍、結果與討論………………………………………………………………74
陸、結論………………………………………………………………………101
柒、參考文獻…………………………………………………………………103
扒、英文摘要…………………………………………………………………11
