臺灣博碩士論文加值系統

　　腸病毒71型是屬於小RNA病毒科的一員，其基因組為一條大小約7.5 kb的正單股RNA，這條RNA被包裹在由VP1到VP4四個蛋白所構成的病毒外殼中，本身沒有外套膜。腸病毒71型大多感染五歲以下的幼童，所造成的症狀涵蓋了輕微的手足口症、咽峽炎，到嚴重的無菌性腦膜炎、腦幹腦炎、急性無力麻痺、小腦性運動失調，甚至是神經性肺水腫。腸病毒71型也在一九九八年在台灣造成大流行，有近十三萬的孩童受到感染，其中有405例為重症患者，最後造成78名孩童的死亡。
　
　　由於腸病毒71型會造成許多神經方面的併發症，所以本研究的目的在於探討腸病毒71型外殼蛋白 (VP1和VP2) 是否具有神經毒性以及其他生物特性。首先利用桿狀病毒表現系統，以攜帶有VP1或VP2基因的重組桿狀病毒感染昆蟲細胞 (Spodoptera frugiperda； Sf-9)，再利用鎳離子親合性管柱純化。實驗結果顯示：VP1蛋白在桿狀病毒表現系統中表現量並不高，而VP2蛋白則是約有70 % 都是inclusion body，爲了獲得較大量的目標蛋白，在實驗中將蛋白表現的平台轉換到大腸桿菌表現系統。在此系統中，以pET系統中的pET21b作為表現載體，另外選了三個不同的菌株作為表現宿主，分別是：BL21(DE3)pLysS strain、BL21-CodonPlus(DE3)-RP strain、BL21-CodonPlus(DE3)-RIL strain來作為表現的宿主。由實驗結果得知，目標蛋白在BL21-CodonPlus(DE3)-RIL中有較高的表現量。在大腸桿菌表現系統中，VP1以可溶性蛋白的方式存在，而VP2仍舊是不可溶蛋白的形式。在表現系統得到大量的蛋白之後，利用鎳離子親合性管柱加以純化，所得到的目標蛋白進行神經毒性測試、趨化性測試以及侵入性測試。在神經毒性測試方面，以不同濃度 (1 µM、3 µM、5 µM) 的目標蛋白 (VP1和VP2) 刺激兩種不同的神經細胞株 (SK-N-SH和SH-SY5Y) 來測試目標蛋白是否具有毒性，實驗結果顯示：這些目標蛋白不論是刺激SK-N-SH或SH-SY5Y，均不會造成神經細胞的死亡。在趨化性測試方面，以不同濃度 (1 µM、3 µM、5 µM) 的目標蛋白 (VP1和VP2) 吸引U937細胞，VP1蛋白可以誘導U937細胞產生趨化性行為，而且不論U937細胞是否有先被VP1處理過，均會隨下方VP1之濃度上升而增加穿透至下方之現象。反之，VP2雖然也能誘導U937細胞產生趨化性行為，但是並沒有隨濃度增加而上升的現象。最後在侵入性測試方面，同樣以不同濃度 (1 µM、3 µM、5 µM) 的VP1或是VP2吸引單核球細胞，結果顯示：無論是VP1或是VP2，均無法明顯誘導單核球細胞產生侵入性行為。由本研究的結果可知：腸病毒71型的外殼蛋白 (VP1和VP2) 雖然對神經母細胞瘤細胞株不會產生神經毒性，也不會造成細胞株的死亡。然而這些外殼蛋白卻可以誘導單核球細胞產生趨化性行為，但卻抑制了侵入性行為的發生。

　Enterovirus 71 (EV71) belongs to Picornaviridae family, its genome is a single-stranded, positive-polarity RNA, approximately 7.5 kb in size. The RNA genome was surrounded by a nonenveloped capsid, which consisted of VP1 to VP4. Infection of EV71 can cause various clinical manifestations, including: hand-foot-and-mouth disease, herpangina, aseptic meningitis, brainstem encephalitis, acute flaccid paralysis, cerebellar ataxia, and pulmonary edema. EV71 caused an outbreak in Taiwan in 1998, nearly 130,000 children were infected, 405 were combined with severe neurological complications, 78 died.

　Because EV71 infection can cause diverse symptoms of CNS, the specific aim of this study is to explore the role of capsid protein (VP1 and VP2) in EV71 infections. At first, baculovirus expression system was used. Recombinant baculovirus which carried the VP1 or VP2 gene infected the Spodoptera frugiperda (Sf-9) cells, then VP1 or VP2 protein was purified via the nickel affinity chromatography. The results showed that VP1 protein could not be expressed in high level, and VP2 protein was expressed in the form of inclusion body. To obtain adequate target proteins, Escherichia coli (E. coli) expression system was used instead. In this system, VP1 or VP2 gene was cloned into pET21b vector, and three different host strains, BL21(DE3)pLysS, BL21-CodonPlus(DE3)-RP, BL21-CodonPlus(DE3)-RIL were analyzed for the VP1 or VP2 expression. The highest expression level was obtained in BL21-CodonPlus(DE3)-RIL strain. The expressed proteins were purified by nickel affinity chromatography and then analyzed their biological properties. In neurotoxicity assay, two neuroblastoma cell lines, SK-N-SH and SH-SY5Y, were pretreated with different doses (1 µM、3 µM、5 µM) of VP1 or VP2 proteins. The results showed that VP1 or VP2 protein can not cause the death of neuroblastoma cell lines. In chemotaxis assay, VP1 can promote chemotaxis behavior of U937 cells in a dose-dependent manner no matter whether U937 cells were pretreated with VP1 or not. On the other hand, VP2 can also promote the chemotaxis behavior, however, dose-dependent manner was not observed. In invasion assay, neither VP1 nor VP2 protein can induce the invasive behavior of peripheral blood monocytes. In conclusion, the capsid proteins (VP1 and VP2) of EV71 did not show neurotoxicity on neuroblastoma cell lines, but they could induce the chemotaxis behavior and inhibit invasive behavior of monocytes.

目錄
中文摘要...................................................................i
英文摘要.................................................................iii
誌謝.......................................................................v
目錄......................................................................vi
圖目錄....................................................................ix
緒論.......................................................................1
腸病毒71型簡史.............................................................1
腸病毒71型的病毒學.........................................................2
腸病毒71型的致病機轉.......................................................4
腸病毒外殼蛋白 (capsid protein) 的研究.....................................6
其他病毒外殼蛋白的研究.....................................................7
神經毒性 (neurotoxicity) 的研究............................................7
趨化作用 (chemotaxis) 的研究..............................................10
侵入性 (invasion) 的研究..................................................13
表現系統的建立............................................................14
研究動機..................................................................16
材料與方法................................................................17
細胞培養、解凍與保存....................................................17
腸病毒71型的培養..........................................................18
萃取病毒的核醣核酸 (ribonucleic acid；RNA)................................18
RT-PCR....................................................................18
洋菜膠電泳................................................................19
DNA片段之回收.............................................................19
限制酶切割處理與純化......................................................19
載體 (vector) 的CIP處理與純化.............................................20
接合反應 (ligation).......................................................20
轉型 (Transformation).....................................................20
目標基因的核酸定序 (sequencing)...........................................21
轉染 (transfection).......................................................21
溶菌斑測試 (plaque assay).................................................22
重組桿狀病毒的小規模量化..................................................22
重組病毒的量化與鑑定......................................................23
重組病毒的鑑定 (基因層面-PCR).............................................24
SDS-PAGE..................................................................25
重組病毒的鑑定 (蛋白質層面-Western blot)..................................25
重組病毒的大規模量化與病毒濃度測定........................................26
以桿狀病毒表現系統表現蛋白................................................26
以大腸桿菌表現系統表現蛋白................................................26
親合性管柱層析............................................................27
神經毒性測試 (Neurotoxicity assay)........................................27
XTT assay.................................................................28
趨化性試驗 (chemotaxis assay).............................................28
侵入性試驗 (Invasion assay)...............................................29
Peripheral blood mononuclear cell (PBMC)分離法............................30
Red blood cell (RBC) lysis buffer.........................................31
結果......................................................................32
VP1和VP2的質體構築、表現與純化..........................................32
桿狀病毒表現系統中目標蛋白 (VP1和VP2) 的表現............................33
大腸桿菌表現系統中目標蛋白 (VP1和VP2) 的表現..............................34
目標蛋白的大量表現和純化..................................................34
神經毒性測試..............................................................35
趨化性 (chemotaxis) 試驗..................................................36
侵入性 (invasion assay) 試驗..............................................37
討論......................................................................38
目標蛋白在不同表現系統中的比較..........................................38
本實驗與文獻中對病毒外殼蛋白的探討之比較................................40
神經毒性測試與實驗模式....................................................41
趨化性和侵入性試驗的探討................................................43
結語....................................................................45
參考文獻..................................................................46
附錄......................................................................87
Appendix 1. 儀器........................................................87
Appendix 2. 藥品及試劑 (組).............................................90
Appendix 3. Media and Broths............................................93
Appendix 4. primers.....................................................98
Appendix 5. Restriction map of pBlueBac4.5/V5-His.......................99
Appendix 6. Restriction map of pET-21b (+).............................100
自述.....................................................................101

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