點帶石斑魚 (Epinephelus coioides)為臺灣具高經濟價值養殖魚之一，目前台灣石斑魚養殖受病原感染問題嚴重，尤其在育苗階段，感染後死亡率極高，造成龐大的經濟損失。視黃酸誘導基因-I (RIG-I)是細胞內識別病毒雙鏈RNA的一種受體，當宿主受到病毒感染時，視黃酸誘導基因-I蛋白產物會大量表現，以介導第一型干擾素 (type I IFNs)抗病毒反應訊息傳導。本研究以點帶石斑魚卵萃取RNA，經RT-PCR增幅可表現視黃酸誘導基因-I調節功能區 (regulatory domain；RD)的cDNA片段，長度為432 bp，將之選殖並建構原核細胞表現重組質體pHis-RIG-I-RD，並在原核系統表現分子量為17 kDa的His-tagged Rig-I-RD融合蛋白 (His-RIG-I-RD)。經大量純化濃縮後，施打於紐西蘭白兔製備多株抗體，並進一步測試此多株抗體之特異性及其力價，西方墨點法結果顯示，於抗體稀釋倍數固定為1:5000的條件下，可偵測到總含量0.01 μg以上之His-tagged Rig-I融合蛋白；於融合蛋白的含量固定為0.02 μg條件下，稀釋倍數為1:25000之抗體仍可有效偵測其蛋白反應。研究中並利用Poly(I:C)模擬病毒核酸以誘導點帶石斑魚頭腎組織表現視黃酸誘導基因-I蛋白，再以所製備抗體偵測其內生性視黃酸誘導基因-I蛋白。西方墨點法結果顯示，此抗體可檢測在點帶石斑魚頭腎組織蛋白萃取物中，由Poly(I:C)刺激誘導產生的視黃酸誘導基因-I蛋白，分子量約為72 kDa。組織免疫切片染色分析結果也顯示，此抗體可在Poly(I:C)誘導的點帶石斑魚頭腎組織冷凍切片中，於黑色素巨噬中心 (melanomacrophage center；MMC)可偵測到較大量視黃酸誘導基因-I蛋白之表現，而在沒有Poly(I:C)誘導的組織切片中，只偵測到微量的視黃酸誘導基因-I蛋白。總結而言，本研究所製備之抗體可做為點帶石斑魚免疫反應中偵測視黃酸誘導基因-I蛋白的表現之試劑，可應用於點帶石斑魚體病毒感染免疫反應及訊號傳遞機制之探討。

Orange-spotted Grouper (Epinephelus coioides) is one of the important economically farmed fish in Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detection of pathogen is critical to prevent epidemic outbreak. Retinoic acid inducible gene-I (RIG-I) is an intracellular receptor recognizing dsRNA virus. Upon dsRNA virus infection, RIG-I is induced and over-expressed in protein level that mediating type I Interferon (IFNs) antiviral signaling. In this study, total RNA extracted from fertilized eggs of Epinephelus coioides was extracted for the reverse transcription-polymerase chain reaction (RT-PCR) amplification of 432 bp cDNA encoding regulatory domain (RD) of RIG-I protein. The amplified cDNA fragment was then cloned into pQE32 for the prokaryotic expression of 17 kDa His-tagged RIG-I (His-RIG-I-RD) fusion protein. His-RIG-I-RD fusion protein was purified for immunization of New Zealand white rabbit to produce antiserum against RIG-I protein. Our Western blot result confirmed the specificity of antiserum for recognizing 0.01 μg His-RIG-I-RD fusion protein at 1:5000 dilution. In addition, 0.02 μg of immunogen His-RIG-I-RD fusion protein can be readily detected at 1:25000 dilution. Furthermore, this antiserum could detect the increase of a 72 kDa protein in lysate prepared from head kidney of dsRNA viral mimic poly(I:C) injected Epinephelus coioides, but not in uninjected control fish. The immunostaining results of frozen head kidney sections also revealed the RIG-I immunoreactivity using this antiserum. RIG-I protein immunoreactivity was prominently increased in melanomacrophage center (MMC) of head kidney from poly(I:C) injected fish compared to that of uninjected control fish. In summary, we have generated a specific antiserum against RIG-I protein of Epinephelus coioides, which could be a useful reagent for further study of Grouper immune response and signal transduction upon virus infection.

頁數
中文摘要 1
Abstract 3
前言 5
實驗目的 16
材料與方法 17
結果 35
討論 42
參考文獻 45
附錄 50
未來與展望 78

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