傳染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)屬於兩段雙股核醣核酸病毒科(Birnaviridae)，為感染經濟性魚貝類之重要水生生物病毒，所以瞭解其致病機轉相當重要。先前研究證實IPNV感染大眼鮭魚胚胎細胞株(CHSE-214)後會引發細胞凋亡，但在整個病毒感染過程中，病毒本身所表達之基因是否有參與其中，尚未清楚。先前本實驗室已初步知道傳染性胰臟壞死病毒其外殼蛋白VP3基因，能誘發斑馬魚上皮肝臟細胞株(ZLE)凋亡及促進(up-regulate)促凋亡基因Bad的表現。由於VP3蛋白於傳染性胰臟壞死病毒所誘發細胞凋亡的角色仍不清楚，因此本論文將更進一步探討，外殼蛋白VP3於傳染性胰臟壞死病毒所誘發宿主細胞凋亡之角色。
首先，利用免疫螢光染色追蹤VP3蛋白之細胞內位置，我們發現當IPNV感染宿主細胞後產生的VP3蛋白將部份跑到粒線體上，我們進一步分離粒線體，並鑑定VP3蛋白是否能跑到粒線體上，其結果與免疫螢光染色一致，尤其在病毒感染之後第9小時更是明顯。同時若於宿主內大量表現作用於粒線體的抗凋亡蛋白Bcl-xL，同樣能有效減緩病毒所造成的細胞凋亡現象。
接著利用antisense RNA方法來阻斷(knockdown)VP3蛋白的表現，便於了解其對宿主細胞存活之影響，分別(1) 偵測細胞凋亡早期marker細胞膜上phosphatidylserine (PS)外翻；(2) 偵測粒線體膜電位之失衡；(3) Bad的表現量之降低；(4) 對病毒效價之降低，其結果一致，當減少VP3蛋白的表現時，能降低胰臟壞死病毒所誘發宿主細胞凋亡與病毒效價。接著更進一步將VP3基因截切為不同片段並接合到綠色螢光蛋白(EGFP)基因，轉染至ZLE細胞中大量表達，發現在VP3基因的第80個到第158個胺基酸序列，可能含有某特定功能區域(functional domain)而具有較強的誘發凋亡能力。
最後為了進一步瞭解VP3如何扮演誘發凋亡角色，我們發現到在病毒感染過程中，VP3蛋白會被磷酸化所修飾。另外我們也發現VP3蛋白本身也具有酪胺酸激酶(tyrosine kinase)功能，藉由處理酪胺酸激酶抑制劑genisten，能有效抑制VP3蛋白的激酶活性並且提升細胞存活率。
本論文針對VP3蛋白凋亡機制之研究，希望能對於瞭解傳染性胰臟壞死病毒致病機制和疾病控制有所幫助。

Infectious Pancreatic Necrosis Virus (IPNV) is one of the widespread fish pathogen and infects many economically important finfish and shellfish. IPNV-infected chinook salmon embryo cells (CHSE-214) could induce cell death through apoptosis. Further, we have found that the submajor capsid protein VP3 can induce apoptosis in ZLE cell line, but its cell death mechanism is still unknown. In this thesis, we further want to know VP3 death mechanism during IPNV infection.
First, we have found that VP3 could target into mitochondria at early-middle replication stage with the immunofluorescence staining assay and Western blot from whole mitochondria. On the other hand, IPNV-induced CHSE-214 cell death through mitochondrial membrane potential loss could be blocked by overexpression of zfBcl-xL. Then, with using of antisense RNA approach for knockdown of VP3 expression during IPNV infection, in the results, (1) can enhance the cell viability, (2) inhibit either PS exposure or mitochondrial membrane potential (MMP) loss, (3) knock down the Bad expression, and (4) reduce the virus titer. Furthermore, VP3 and a series truncated forms were constructed for expression in ZLE cells. We found that the VP3 death motif may be located between 80-158 amino residues. Then, we found the VP3 may have a tyrosine kinase activity by immunoprecipitation assay between 3 hour and 12 hour post-infection and VP3 may reserve as a substrate for either tyrosine or serine/threonine kinases, but their functions are still further examined.
Taken our results suggest that VP3 may play a crucial role on pro-apoptotic function, which may up-regulate the pro-apoptotic gene Bad and induces the loss of MMP in CHSE-214 cells with IPNV infection. This finding may thus provide an important insight into the understanding of IPNV pathogenesis and disease control.

戴君如，以傳染性胰臟壞死病毒之VP3反意核醣核酸表現抑制本病毒所誘發之細胞凋亡。台灣大學漁業科學研究所碩士論文，台北，2000。
邱謙禮，傳染性胰臟壞死病毒外殼蛋白VP3造成斑馬魚肝臟細胞株凋亡分子機制之研究。成功大學生物科技研究所碩士論文，台南，2006。
陳栢均，傳染性胰臟壞死病毒造成宿主細胞大眼鮭魚胚胎細胞株因粒線體膜電位消失而凋亡之研究。成功大學生物科技研究所碩士論文，台南，2007。
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