臺灣博碩士論文加值系統

　　細胞常藉由調控程式化凋亡來清除被病毒感染之宿主細胞。神經壞死病毒感染宿主細胞時，已知可引起細胞凋亡，但病毒如何促進或抑制宿主凋亡之機制尚不清楚。利用西方轉印免疫實驗中，首次發現在病毒感染後24 小時，B2 蛋白質(8.3 KD)開始持續表現。為探討此神經壞死病毒B2 蛋白所扮演之功能，我們從病毒次基因體RNA3 中，選殖出此非結構性蛋白基因B2。B2基因全長含有228 個核苷酸，可轉譯出75 個胺基酸。若將B2基因接到黃色螢光基因(EYFP)後面形成融合基因，並轉染至細胞內，表現B2 蛋白會進入粒線體內。如果將B2基因短暫的表現於石斑魚肝臟細胞株(GL-av)內，可誘發細胞凋亡。同時，當我們短暫表現B2 基因與抗斑馬魚凋亡基因Bcl-xL 及Mcl-1a 時，則可減緩B2所誘發之細胞凋亡現象。此外，當利用基因knockdownz 方式之小片段干擾性RNA(siRNA)可有效阻斷B2 蛋白之表現，則可減少細胞凋亡及減緩病毒複製。由本論文之研究，證實B2 蛋白是屬早期表現之粒線體蛋白。其功能則與誘發宿主細胞凋亡及病毒複製與蛋白表現有密切相關。希望此研究之發現，能對防治或預防神經壞死病毒所誘發之疾病有所助益。

　　Apoptosis is a genetically-controlled preprogrammed event which eliminates cells during viral infection with host cells. Recently,betanodavirus-induced host cell death through apoptosis have been reported, but virus how to involved in regulate the host cell death is still a few understood. We found that B2 protein was expression occurred at 24 h post-infection (p.i.). So, we interest to know its function, then B2 gene be cloned and characterized was done. The betanodavirus TN1 strain B2 cDNA is 228 nucleotides long, encoding a polypeptide of 75 amino acids that protein size about 8.3 kDa in grouper liver (GL-av) cells. EYFP as a reporter gene was used for tracing B2 protein that also found localized in mitochondria in GL-av cell after 24 h p.i. When overexpression of B2 gene that significantly induces GL-av apoptotic cell death, but also prevents cell death by extra expression of Bcl-2 member Bcl-XL and Mcl-1a, which were cloned from zebrafish. Moreover, siRNA approaches are used for knockdowned B2 expression in transient and stable expression cell line in GL-av cell, which can enhances the cell viability up to 30% at 24 h p.i. and reduces the lose of mitochondria membrane potential during NNV infection up to 20% from 24 h to 72 h p.i., respectively. Taken results suggest that B2 is an early expression gene which may play an important role on either induces the host post-apoptotic necrosis or may regulate the viral replication. This finding may thus provide an important insight into the control or prevention of nodavirus-induced diseases.

目錄
中文摘要 II
英文摘要 III
誌謝 IV
目錄 V
表圖目錄 VII
一、緒論
(一) 石斑魚之基本介紹 1
(二) 神經壞死病毒之基本特性 2
(三) 病毒引起宿主細胞凋亡之研究 5
(四) 核醣核酸干擾術 ( RNA interference ) 9
(五) 目的 12
二、材料與方法
A. 材料 13
B. 儀器 22
C. 方法 25
三、結果
(一) NNV 感染GL-av 細胞後病毒蛋白表現情形 45
(二) 病毒基因B2 基因選殖及質體構築 45
(三) B2 基因功能分析 47
(四) B2 基因誘發細胞凋亡之分子機制 51
(五) 利用siRNA 系統阻斷B2 基因表現 52
(六) 神經壞死病毒感染B2 si RNA 之穩定表現細胞株 55
四、討論與展望
(一) 魚類細胞株基因轉染效率低 57
(二) B2 蛋白位置及功能之相關性 58
(三) siRNA 設計之位置及使用 59
(四) 展望 61
五、參考文獻 62
六、附錄 69
七、自述 103

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