點帶石斑魚 (Epinephelus coioides) 為台灣重要的養殖經濟魚種，但在養殖過程中常常因為疾病的感染而造成過多損失，因此研究魚體遭遇病原體時的反應機制是非常必須且急迫的。先天免疫反應作為抵抗疾病的第一道防線，有許多細胞激素參與其中，例如腫瘤壞死因子-α (tumor necrosis factor alpha, TNF-α) 是初期炎症反應的重要分子之一，能夠刺激免疫細胞並活化後續一連串的免疫反應。
目前在部分的魚類發現具有一種以上的TNF-α蛋白，這與我們所熟知的哺乳動物僅有一個TNF-α蛋白不同。在本研究中，成功選殖到與過去所發現的點帶石斑魚TNF-α (稱為TNF-α1) 不同的另一個TNF-α基因，命名為TNF-α2。分析點帶石斑魚的TNF-α2基因，其mRNA長度為1510個鹼基對，推測產生240個胺基酸。TNF-α1與TNF-α2在開放閱讀框架 (open reading frame, ORF) 區域核苷酸序列相似度為52 % ，而胺基酸組成相似度則為35 % 。觀察不同臟器在LPS刺激下的TNF-α表現量，發現TNF-α1與TNF-α2具有不同的表現pattern，TNF-α2的表現更為全面性；且在觀察魚苗發育階段時，發現TNF-α1與TNF-α2的mRNA表現量似乎在水中弧菌數升高時，皆會受到刺激而有上升的現象，但TNF-α1與TNF-α2的表現趨勢不一致。這些結果支持此二TNF-α分子可能具有不同的功能。由於在他人研究中對魚類相異之TNF-α分子其功能性與調控表現僅有初步的探討，因此本研究將藉由所選殖到的TNF-α1與TNF-α2啟動子區域，利用點帶石斑魚細胞繼續分析在不同狀況下此二基因的表現及調控；另一方面則表現rTNF-α1與rTNF-α2免疫兔子取得多株抗體，從蛋白質層面上研究兩者的差異性，以了解兩種不同的TNF-α在免疫反應上的角色及關係。

Epinephelus coioides (E. coioides, orange-spotted grouper) is an important cultivated fish that has value of economy in Taiwan. Infecting with diseases was causing huge loss in aquatic industry. To uncover the response of fish that have encountered pathogens is necessary and urgent.
Many cytokines involved in innate immunity and work as the communication and regulation of the immune system. Tumor necrosis factor alpha (TNF-α) is an important molecule, which was secreted in primary inflammatory reaction. TNF-α can activate the immunocytes and the innate immune network system. There are more than one of TNF-α molecular were found in some fish, it is quite different with human and vertebrate animal we usually known. In this study, a novel TNF-α (TNF-α2) gene of E. coioides has been cloned. The length of mRNA is 1510 bp and the predicted protein has 240 amino acids. The identity between the ORF region of TNF-α1 and TNF-α2 was 52 % in uncleartide and 35 % in amino acid sequence. In order to identify the mRNA expression in various organs, the groupers inject intraperitoneally with LPS (40 mg / kg of body weight). Then organs are collected after 24 h and evaluated the expression of TNF-α mRNA. The expressional pattern of TNF-α1 and TNF-α2 are dissimilar. In larval stage, all two TNF-α perhaps are activatable by raise of Vibrio. Also these two TNF-α have different expression pattern under stimulation of bacteria. These results show that two TNF-α molecules may have different functions. However, the functions, distribution in different tissues, and regulation by different pathogens or stimulators of these two TNF molecules were not sufficient researched. In order to understand the difference between TNF-α1 and TNF-α2 in regulation mechanism, the promoter region of TNF-α genes have been cloned and analyzed. The transcription factor binding sites of TNF-α1 and TNF-α2 are dissimilar. In the future, the deletion assay of promoter region will proceed in cell line and the polyclonal antibody of TNF-α will be prepared to detect TNF-α expression in protein level. These can help us understand the roles of TNF-α1 and TNF-α2 in immune response.

邵廣昭 台灣魚類資料庫 網路電子版 version 2009/1 http://fishdb.sinica.edu.tw, (2010-7-25)

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