|Abstract: ||石斑魚是台灣重要的經濟海水養殖魚類，虹彩病毒 (iridoviruses) 是其重要病原體之一，因此研究石斑魚的抗虹彩病毒免疫防禦機制是非常重要的。本實驗室先前的研究發現，點帶石斑魚 (Epinephelus coioides) 被石斑魚虹彩病毒 (grouper iridovirus, GIV) 感染後，脾臟中的CC趨化素基因EcSCYA102、EcSCYA103、EcSCYA116及EcSCYA118的表現會顯著上升，代表這四個趨化素可能與抗GIV有關。本碩士論文接續研究這四個基因，分成兩部分：基因表現分析及其作為DNA疫苗佐劑的效能測試。首先我們利用Real time PCR技術檢測了這四個基因在點帶石斑魚的肝臟、頭腎、腸、鰓和脾臟的基因表現情形。無處理魚隻體內，這四個基因在肝臟的表現量都是最低的，而EcSCYA116和EcSCYA118在肝臟以外的其他器官的表現量差異不大，但EcSCYA102和EcSCYA103則具有相當明顯的基因表現偏好性，分別在腸道和鰓有最高的表現量。在GIV感染後，這四個基因的表現量都會顯著上升，其中EcSCYA118能在最多的器官中被誘導表現；在LPS刺激下，只有EcSCYA116/118會被強烈誘導表現，表示這兩種趨化素可能具有對細菌的防禦能力；而以Poly I:C刺激時，EcSCYA102/103/116表現量均會顯著上升，其中EcSCYA102對Poly I:C最為敏感。為了測試這四個基因是否具有DNA疫苗佐劑的效能，我們構築了重組質體pcDNA3-EcSCYA102、pcDNA3-EcSCYA103、pcDNA3-EcSCYA116及pcDNA3-EcSCYA118；另以GIV 主要外殼蛋白MCP (major capsid protein) 基因片段 作為DNA疫苗，構築重組質體pcDNA3-GIV MCP。將這些重組載體DNA注射入魚體肌肉後，以酵素連結免疫吸附法 (enzyme-linked immunosorbent assay，ELISA) 進行抗GIV MCP抗體效價分析。結果顯示疫苗組與空載體組以及疫苗組與疫苗添加佐劑組並沒有顯著差異，但疫苗添加EcSCYA103/116組及疫苗添加EcSCYA102/118組分別在注射後第14天和第21天與空載體組有顯著差異，代表這四個趨化素確實有提升抗體效價的能力。綜上述的結果，EcSCYA102/103/116/118具有成為DNA疫苗佐劑的發展性，且EcSCYA118可能是較有潛力的選擇。|
Grouper is an important marine aquaculture fish in Taiwan, and iridovirus is the important pathogen for grouper. Therefore, it is very important to understand the immune responses in grouper triggering by iridovirus infection. In our previous study, we found that after grouper iridovirus (GIV) infection, the expression of several CC chemokine genes, EcSCYA102, EcSCYA103, EcSCYA116 and EcSCYA118, were markedly increased in the spleen of orange-spotted grouper (Epinephelus coioides), suggesting that these CC chemokines might involve in immunity against GIV infection. This Master Thesis continued the study of these four CC chemokine genes, with two purposes: analysis of their gene expression pattern and investigation on the possib ility of usig them as DNA vaccine adjuvant. For gene expression analysis, we used Real time PCR to detect the expression of these four genes in liver, head kidney, intestine, gill and spleen. In untreated fish, all the four CC chemokine genes had lowest expression level in liver; excluding liver, the various organs had similar expression levels of EcSCYA116 and EcSCYA118; both EcSCYA102 and EcSCYA103 exhibited preferential expression in intestine and gill, respectively. GIV infection could significantly induce the expression of all the four genes, and among them, EcSCYA118 could be induced in most assayed organs. Expressions of EcSCYA116 and EcSCYA118 were induced by LPS, suggesting that both might be related to resistance against bacteria. EcSCYA102/103/116 could be significantly up-regulated by Poly I:C injection and amon them, EcSCYA102 was most sensitive to Poly I:C. To explore the possibility of using the four CC chemokine genes as DNA vaccine adjuvant, expression plasmids pcDNA3-EcSCYA102/103/116/118 were constructed; in terms of DNA vaccine, GIV MCP (major capsid protein) gene fragment was used and constructed as pcDNA3-GIV MCP. These recombinant expression plasmids were intramusclarly injected into the grouper and then the levels of anti-GIV MCP antibodies in fish serum were assayed by enzyme-linked immunosorbent assay (ELISA). The results showed MCP group had no significant difference with either empty plasmid group or MCP＋adjuvant group. However, compared with empty vector group, MCP＋EcSCYA103/116 groups and MCP＋EcSCYA102/118 groups had significantly elevated antibody titer at 14 and 21 days post-injection, repectively. The result suggested that these chemokines indeed have the ability to increase antibody titer and might have the potentiality to be used as DNA vaccine adjuvant, and EcSCYA118 could be a more promising option.