脊椎動物的干擾素(interferon)防禦機制為抵抗病毒的第一道防線，它藉由啟動合成下游蛋白來抑制病毒的複製，至少有三種以上的抗病毒蛋白受到干擾素的調控，而Mx蛋白就屬其中一種，Mx蛋白對於病毒複製的抑制特別是在RNA病毒方面被研究的相當透徹。點帶石斑魚(Epinephelus coioides)，台灣的經濟養殖魚類之ㄧ，在整個養殖過程中幼魚受到神經壞死病毒(nervous necrosis virus)感染死亡情形相當嚴重，但在成魚則相對的沒那麼嚴重，因此點帶石斑魚似乎有一套防禦機制對抗神經壞死病毒的感染。在之前的工作，點帶石斑魚的Mx cDNA已被選殖，全長1881bp並且轉譯出626個胺基酸，在結構上來看石斑魚Mx蛋白(gMx)跟哺乳類一樣都屬大型GTP結合蛋白。這裡我們探討石斑魚泳鰭細胞(GF-1)及石斑魚肝臟細胞(GL-a)皆在受到干擾素誘導物poly[I:C]處理後，啟動內生性免疫反應防止細胞受NNV感染死亡現象。並以GL-a細胞做免疫螢光染色，以及在GF-1細胞transient表現gMx-GFP蛋白的方式，探討gMx蛋白在細胞內的駐留位置，發現Mx蛋白在感染NNV後有聚集在內質網的現象，並且跟神經壞死病毒外殼蛋白有部分重疊現象。另以MTT測定持續表現Mx蛋白之GF-1細胞感染NNV後，有提高細胞存活率，推測Mx蛋白應參與內生性的防禦NNV機制。在gMx跟nodavirus蛋白之間的關係方面，以GST-pull down及Far western blot的方式，測試gMx蛋白質的功能片段GTP-ase domain、CID(central interactive domain)、Leucine zipper domain是否會跟NNV 外殼蛋白有結合的現象，結果顯示gMx的CID(335-503 aa)及L/Z domain(556-626 aa)可跟NNV外殼蛋白結合。綜和以上各點，Mx蛋白在石斑魚可能參與內生性免疫保護機制，而此機制可能透過跟NNV外殼蛋白結合，阻礙病毒組裝有關，因此gMx蛋白將可被利用在石斑魚抵抗神經壞死病毒的一大利器。

　The defense system of interferon(IFN) in vertebrate provides an initial line of defense against viral infection by inducing the synthesis of proteins that inhibit virus replication. At least three different proteins mediate the antiviral response and one of them, Mx protein, specifically inhibits the replication of wide range of RNA virus has been well established. Grouper(Epinephelus coioides)is one of the economical fish in Taiwan. Because the virus infection in adult red-spotted grouper is often light and unrecognized, grouper should have an efficient defense system against to nodaviruses. In the recent work, a homolog Mx (“OsgMx”) gene from orange-spotted grouper (Epinephelus coioides) had been cloned and sequenced. It contains an 1881 bp open reading frame region and encodes a protein with 626 amino acids. On protein structure, the grouper Mx protein is similar to the superfamily of large GTP-binding proteins. In this study, we found that the gMx could be expressed in grouper cell lines, GF-1(grouper fin cell) and GL-a(grouper liver cell), after treated with interferon-inducer poly [I:C] and it could prevent cell from death. The localization of gMx protein was determined by transient expression of gMx-GFP in GF1 cell line and detection endogenous gMx protein in GLa cell by immunofluorescence. The gMx protein was located in cytoplasm and assembles in endoplasmic reticulum after nodavirus infected. A cell transfection assay was used to examine the protection of the gMx cDNA clones against infection of NNV. The gMx would be a part of innat immunoresponses defence NNV. Furthermore, GST-Mx fusion proteins, which contain the major domain of GTP binding domain, central interactive domain (CID; 335-503 aa) and Leucine zipper domain(L/Z; 556-626 aa) were expressed to demonstrate that gMx protein could bind to nodavirus coat protein by GST pull-down and Far western blots. The CID and L/Z domain were the majar domains to associate with NNV coat protein. In conclusion, gMx protein in grouper maybe function as a part of protection system, which was related to nodavirus coat protein and to interfere with virus assembling processes in the cellular context against nodavirus, and prevents cell from death. In the future, gMx protein will be applied as a grouper antivirus agent on young grouper.

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