臺灣博碩士論文加值系統

研究指出水溫升高會降低蝦類受白點症病毒感染之死亡率，但未深入探討其影響機制。本研究以草蝦 (Penaeus monodon) 為生物性樣材，分析白點症病毒於25或32℃水溫感染草蝦時，病毒複製、基因轉錄與轉譯之差異。根據病毒DNA檢測之結果顯示，溫度為25℃時，蝦體於12 hpi.（hour post-infection）呈現重度感染狀態，病毒出現大量複製，而於32℃時，直至48 hpi.仍為輕度感染，病毒少量複製狀態，因此推論病毒於32℃下不能有效進行複製增殖。另針對數個病毒基因之轉錄表現時序分析，在25與32℃下，白點症病毒潛伏性相關基因wssv194於2 ~ 48 hpi.時為持續穩定表現；在25℃下，極早期基因ie1、ie2以及早期表現基因-- DNA聚合酶 (DNA polymerase)，在2 ~ 48 hpi.時基因表現隨感染後時序的增加而上升，12 ~ 48 hpi.時上升最為明顯，但32℃時，上述基因在2 hpi.時即有高表現量，且至48 hpi.皆呈現持續穩定表現，顯示在32℃時基因轉錄作用提昇。若以西方轉印法偵測病毒蛋白質表現，則顯示感染溫度於25℃下，24 ~ 48 hpi.時可偵測到IE1與VP28蛋白質存在，而32℃下則至48 hpi.皆未能偵測到這些病毒蛋白質之表現，依據過去之研究結果，推論此現象與磷酸化的信號傳導子和轉錄激酶 (signal transducer and activators of transcription, STATs) 有關，因此ie1於32℃時，基因表現時序性提前升高，可能因為未感染病毒之蝦體於32℃養殖下，具較多活化態STATs造成。綜合以上實驗，本研究亦發現在32℃下草蝦感染病毒後，醛脫氫酶 （NAD-dependent aldehyde dehydrogenases） 與蛋白質降解酶 （Proteasome alpha 4 subunit）蛋白質量明顯上升，是否因蛋白質降解酶相關蛋白表現量上升，而促進蛋白質降解之分子機制尚未釐清，仍需待進一步實驗證明。

Hyperthermia promotes survival of the WSSV-infected shrimps and the underlying mechanisms remain unknown. In the present study, we investigated the effects of hyperthermia (25 vs. 32℃) on WSSV gene expression and genome replication in Penaeus monodon shrimp. By using IQ2000 kits, we found that high level of WSSV genome could be detected as early as 12 hours post infection (hpi.) at 25℃, whereas low amount of WSSV genome became detectable until 48 hpi. at 32℃. Temporal analyses of the transcription of several WSSV genes showed that although high infection temperature did not affect the transcription pattern of a latency-associated gene (wssv194), the patterns of two immediate early genes (ie1 and ie2) and the early gene dna polymerase (dna pol) changed dramatically. At 25℃, the expression level of ie1, ie2 and dna pol was low at 2 – 8 hpi., and gradually increased at 12 – 48 hpi. At 32℃, the expression of these three genes surged rapidly to reach a high level by 2 hpi. and then maintained at a constant level until the end of the analysis. The expression of two viral proteins, IE1 and VP28, was investigated using Western blot analysis, and it showed that at 25℃ both proteins were detected at 24 and 48 hpi., but at 32℃, neither proteins were identified. Based on these studies, we concluded that at high infection temperature (32℃), WSSV’s transcription activity was aberrantly high, its replication was blocked and the amount of viral proteins was quite low. STATs up-regulates ie1 gene expression. In the present study, Western blot analysis showed that in normal shrimp, hyperthermia activated STATs, and this might explain in part how ie1 transcription was induced by hyperthermia. Lastly, the protein profile changes in WSSV-infected shrimp induced by hyperthermia were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Two shrimp proteins, NAD-dependent aldehyde dehydrogenases and proteasome alpha 4 subunit, were upregulated significantly. The increased expression of a proteasome-related protein suggested that in WSSV-infected shrimp, hyperthermia might enhance the protein degradation pathway to down-regulate viral protein expression.

前言………………………………………………………………1
材料與方法…………………………………………………… 13
1.白點症病毒株與其病毒液之製備………………………… 13
2.人工注射感染試驗與草蝦組織檢體之收取……………… 13
3.檢體之DNA萃取與檢體感染前後之白點症病毒檢測方法…14
4.檢體之RNA萃取………………………………………………15
5.反轉錄酶—聚合酶鏈反應………………………………… 16
6.檢體全蛋白之萃取與蛋白質定量………………………… 17
7.西方轉印法………………………………………………… 17
8.細胞核與細胞質蛋白質分離萃取………………………… 19
9.蛋白質二維電泳分析……………………………………… 19
10.蛋白質膠體內水解與蛋白質身分鑑定……………………21
結果…………………………………………………………… 22
1.溫度對白點症病毒基因體複製的影響…………………… 22
2.溫度對白點症病毒基因轉錄表現的影響………………… 23
3.溫度對磷酸化的信號傳導子和轉錄激酶 (Signal Transducer and Activators of Transcription, STATs) 之影響…… 25
4.病毒蛋白質時序性表現差異分析………………………… 26
5.草蝦鰓組織不同溫度感染病毒後全蛋白二維電泳分析表現之差異……………………………………………………………… 26
討論…………………………………………………………… 28
參考文獻……………………………………………………… 39
圖表及附錄…………………………………………………… 47

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