點帶石斑魚(Epinephelus coioides 俗稱青斑)由於肉質鮮美，為台灣重要養殖經濟魚種之一。但在養殖的過程中常因為疾病的侵襲而造成損失，魚類疾病的預防有賴於成熟且具功能的免疫系統。但由於魚類為體外受精的動物，故其魚苗發育時期皆暴露於環境之中，比胎生動物更容易受到疾病病原的侵襲。故魚苗時期免疫系統的發育，功能性的探討，以及與病原間的關係遂成為研究預防魚苗疾病及防疫上的重要課題。腫瘤壞死因子(TNF-α)為引起發炎反應最初且最重要的因子之一，主要為誘發炎症反應以及刺激免疫系胞活動所需。故可視為魚隻免疫系統接受外來病原刺激後初期免疫反應的指標物質。在許多魚種中TNF-α基因已被成功的選殖。但根據目前已知物種的TNF-α基因比對結果，發現魚類與哺乳動物的TNF-α基因相似度僅達20-50 %，魚類間的TNF-α相似度則依親源的遠近有著60-90 %之相似度。這個存在的岐異性也造成相關研究的困難。本研究的目地在於選殖點帶石斑魚TNF-α之基因，及分析這個基因在魚苗胚胎時的表現情形。我們成功的選殖到點帶石斑魚TNF-α之基因，基因全長為1516 bp，分為4個exon及3個intron。推定可以產生254個胺基酸，分子量約為27 kDa。推定的胺基酸中包括TNF family signature, transmembrane domain, TACE酵素切位及兩個與結構構型相關的cystein 胺基酸。由胺基酸組成序列上證明其為TNF-α分子。我們進一步依據點帶石斑魚TNF-α基因設計特異性引子。以自石斑魚苗養殖場採集到的魚苗樣本所萃取之mRNA 進行定量反轉錄real time PCR。結果發現魚苗體內TNF-α之基因之mRNA量自第19日後產量有明顯上升的跡象。希望這些研究的成果有助於提供石斑魚魚苗時期相關cytokine表現以及免疫系統的發生更進一步的資訊，以作為魚類魚苗階段使用疫苗或是免疫促進劑以防治疾病時的參考。

Epinephelus coioides (E. coioides, orange-spotted grouper) is an important farmed fish in Taiwan; there is a paramount problem of raring fish is pathogen infection, especially it occurs at larval stage. Fish larvae were not able to against the pathogen invasion because its immune system was incompletely developed. Innate immune response is the first line to defense pathogens invasion by inducing inflammatory reaction, numerous of mediator and immunocytes are intervening in it, TNF-α is the most important cytokines and is secreted in the earliest inflammation reaction, TNF-α gene was found in some of bony fish; however, the gene of most fish is still unknown. In this study, a TNF-α gene of E. coioides was cloned, the gene was 1516 base pairs (bp) long with 4 exons and 3 introns. The length of open reading frame is 762 bp encoding 253 amino acids with the molecular weight of putative protein was about 27 kDa, the TNF family signature, transmembrane domain, TACE cutting site and two cysteins residues which involved in tertiary structure formation were predicted. Subsequently, the TNF-α mRNA expression level in ontogeny of fish and different organs was evaluated by quantitative reverse transcriptase real time PCR (qRT-PCR). The result shown the expression level of TNF-α mRNA raised after 19 dpf of fries. It indicated that the innate immune system was activated at that stage. The development and functions of immunity on larval fish will be comprehended in this study expectantly, furthermore, designing a proper and efficient strategy to prevent infection of larval stage of fish form the information of this study.

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