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| 研究生： |
林浩平 Lin, Hao-Ping |
|---|---|
| 論文名稱： |
點帶石斑魚趨化素接受器CXCR4之功能分析 Functional Analysis of the Chemokine Receptor CXCR4 in Orange-Spotted Grouper (Epinephelus coioides) |
| 指導教授： |
陳宗嶽
Chen, Tzong-Yueh |
| 學位類別： |
碩士 Master |
| 系所名稱： |
生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology |
| 論文出版年： | 2013 |
| 畢業學年度： | 101 |
| 語文別： | 中文 |
| 論文頁數： | 104 |
| 中文關鍵詞： | 點帶石斑魚 、神經壞死病毒 、趨化素 、趨化素接受器 、CXCR4 、CXCL12 |
| 外文關鍵詞： | grouper, nervous necrosis virus, chemokine, chemokine receptor, CXCR4, CXCL12 |
| 相關次數： | 點閱：99 下載：1 |
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點帶石斑魚 (Epinephelus coioides) 是台灣最重要的海水養殖魚種，在高密度養殖過程中易受病原的威脅，尤其是魚苗時期易感染的神經壞死病毒 (nervous necrosis virus, NNV) 常造成嚴重的經濟損失。本研究室先前已成功選殖出點帶石斑魚趨化素接受器 CXCR4 基因 (gCXCR4)，研究結果指出 gCXCR4除了在免疫反應上扮演重要角色外，亦參與在石斑魚早期生長方面的調控。為了瞭解 gCXCR4 在病原感染下之功能與調控，本研究主要分為三個部分做探討。第一部分針對 gCXCR4 和 NNV 之間的調控功能進行分析，藉由免疫組織螢光染色觀察感染NNV後的點帶石斑魚幼魚其 gCXCR4 在組織上的表現情形及位置，發現 gCXCR4 和 NNV 在組織上的表現有著高度的雷同性，經 GST pull-down 以及 Far western 實驗結果証明 gCXCR4 和 NNV 之coat protein (NNV-CP) 彼此間具交互作用。第二部分則以石斑魚鰭細胞株 (GF-1) 觀察點帶石斑魚CXCR4 蛋白質表現位置與特性。利用細胞骨架 f-actin 染劑可進一步觀察到無論 GF-1 細胞中大量表現 gCXCR4 或感染神經壞死病毒，皆會造成細胞型態改變，於細胞膜上產生了許多纖毛狀的構造，且兩者表現會從細胞質轉移至細胞核甚至是核仁之中。第三部分則為選殖及表現點帶石斑魚趨化素 CXCL12 (gCXCL12) 基因，並進行特性分析。該基因之全長序列已成功選殖，其 ORF 為 297 個核苷酸，並可轉譯出 98 個胺基酸。利用RT-PCR 和 Real-time RT-PCR 偵測 gCXCL12 在各組織之間的表現情形，發現 gCXCL12 在頭腎組織中表現量最高。而在攻毒實驗方面，證實 gCXCL12 基因在注射神經壞死病毒後的第 24 小時具最高表現量，於第 48 個小時發現 CXCR4 表現量亦隨之提升。根據本研究成果顯示 gCXCR4/gCXCL12 調控路徑和 NNV-CP 間的作用機制存在著密切關係，可能對於防治 NNV 造成石斑魚養殖業所帶來之危害有所助益，並可提高養殖經濟效益。
Orange-spotted grouper (Epinephelus coioides) is the most important economical cultivate fish in Taiwan coast, but it always threatened by pathogens during intensive cultivation, especially, the nervous necrosis virus (NNV) infection causes massive mortality in grouper larvae. In order to understand the function and regulation of gCXCR4 under NNV infection, the purpose of the present study contains three portions. The first part is evaluating the relationship between NNV and gCXCR4. Immunohisto- fluorescence staining revealed that gCXCR4 was colocalized with the coat protein of NNV (NNV-CP). gCXCR4 and NNV-CP that show robust interactions in GST-pull-down experiments and far western blotting. The second part, cell culture experiments comparing with f-actin staining indicated that the overexpressed gCXCR4 protein not only distributed to cell membrane and cytoplasm but also the nucleus and nucleolus, and result in cell morphology change. The third prat is the molecular cloning and characterization of orange-spotted grouper CXCL12. The open reading frame of gCXCL12 contained 98 amino acid residues with an estimated molecule mass of 11.27 kDa. RT-PCR and Real-time RT-PCR revealed that gCXCL12 presented abundantly in head kidney. Virus challenge experiment indicated that gCXCL12 were 19.5-folds induced at 24h after viral challenge and gCXCR4 expression level were 541.1-folds increased at 48h after viral challenge subsequently. Furthermore, researching on the mechanism between gCXCR4/gCXCL12 regulation pathway in oranged-spotted groupers and NNV infection may help prevent grouper from NNV infection and promote the economic efficient in aquaculture industry.
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校內：2015-02-04公開校外：不公開