點帶石斑魚 CD9 基因啟動子之活性與特性及添加 poly(I:C) 與感染石斑魚虹彩病毒的表現分析

Activity and Characterization of Orange-spotted Grouper (Epinephelus coioides) CD9 Promoter, and Its Expression Analysis in Response to Poly(I:C) and GIV Infection.

劉千維(Chien-Wei Liu)

指導教授 ： 張繼堯

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01575

摘要

石斑魚是台灣水產養殖重要魚種，具高經濟價值，然其幼苗成長時期易受石斑魚虹彩病毒 (grouper Iridovirus, GIV) 感染而大量死亡，故本實驗室以易於養成之點帶石斑魚 (Epinephelus coioides) 為材料，研究石斑魚與GIV之交互作用。2012年吳與劉於本實驗室對點帶石斑魚施打poly(I:C)、GIV及LPS，發現poly(I:C) 及GIV注射後，CD9表現模式與其他干擾素 (interferon, IFN) 相關之基因相仿。IFN在病毒感染後，扮演先天性免疫與後天性免疫間重要的橋樑，影響抗病反應，但CD9的角色是個謎，2012年楊取得點帶石斑魚CD9 cDNA全長，並發現主要表現在免疫器官上，故希望取得CD9啟動子以了解與GIV之關係。
　　根據本實驗室所獲得之點帶石斑魚CD9 cDNA全長，利用染色體步移技術 (Genome Walking)，獲得約1.2 Kb及2.6 Kb的CD9啟動子 (promoter)，經定序並分析其轉錄因子結合位 (transcription factor binding site) 後，預測CD9啟動子可能含有AP-1、ApoD、C/EBP、NF-κB、TNF-α-Y-BOX、IRF1和IRF2的結合位。透過冷光酵素的測定 (Luciferase Assay) ，得知在GK (grouper kidney) 細胞及GB (grouper brain) 細胞，1.2 Kb CD9 有一定量表現，2.6 Kb CD9近乎不表現，且活性皆不受不同濃度poly(I:C) 的影響。藉由GK細胞中CD9啟動子的刪除實驗得知，在1.2 Kb及2.6 Kb 間的CD9啟動子可能含有抑制調控因子的結合位；1.2 Kb CD9在相對靠近轉錄起點端的124 bp及400 bp至679 bp之間可能有關鍵影響CD9表現之結合位。不同大小的CD9啟動子，在GK細胞均不受不同濃度poly(I:C) 的影響，感染GIV後，可能受抑制而近乎不表現CD9。透過NF-κB之次單元c-rel於GK細胞的共同轉染，可促進CD9啟動子的調控。以RT-PCR測定之內生性CD9在GK細胞不同濃度poly(I:C)下不同時間點的表現均無差異。自點帶石斑魚活體的頭腎 (Head Kidney, HK) 及脾臟 (Spleen) 組織純化出的白血球細胞，添加poly(I:C) 後，以RT-PCR測定，發現CD9在HK及Spleen白血球細胞不同的時間點均沒有差異，然而頭腎及脾臟白血球細胞中的IFNa與Mx都會受到poly(I:C) 的刺激而提高表現量，並在達到高峰後降低表現。
　　綜合本研究結果，認為CD9基因的調控可能與NF-κB有關，且可能受到GIV感染而受到抑制，然而CD9的表現是否還可能與其他因子有關及在GIV感染時扮演什麼樣的角色有待進一步證實。

關鍵字

石斑魚虹彩病毒 ； 點帶石斑魚 ； 啟動子 ； 冷光酵素測定

並列摘要

High economic benefits make groupers important aquacultural species in Taiwan. However, the disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Therefore we take Epinephelus coioides which easily raised as our model to study the interactions between groupers and GIV. In 2012, Liu and Wu injected poly(I:C), GIV and LPS into orange-spotted groupers and discovered the expression pattern of CD9 resembling to those genes associated with interferon (IFN). IFN influencing antiviral effects are important bridges between innate immunity and adaptive immunity, but the role of CD9 remains unknown. We gained the total length of CD9 cDNA from orange-spotted grouper (Yang, 2012), finding that CD9 mainly expressed in immune organs. Thus, we hope to get CD9 promoter for further analysis to realize its relationship to GIV. According to the total length of CD9 cDNA gained, we obtained 1.2 Kb and 2.6 Kb CD9 promoters from genome walking. Through analysis of sequence and transcription factor binding sites, we predicted that CD9 promoters might contain binding sites of AP-1, ApoD, C/EBP, NF-κB, TNF-α-Y-BOX, IRF1 and IRF2. By luciferase assay, we found that 1.2 Kb CD9 prmoter but not 2.6 Kb CD9 promoter regularly expressed in
GK cells and GB cells, and their activities weren't affected by different concentration of poly(I:C). In deletion analysis of CD9 promoter in GK cells, 2.6 Kb CD9 promoter might have negative regulatory factor binding sites. Some binding sites located in 124 bp near the side close to transcription initiation point and the part between 400 bp and 679 bp of CD9 promoter might play a key role to affect expression. CD9 promoters with different lengths were also not affected by various concentration of poly(I:C), but the infection of GIV inhibit CD9 promoters, causing extremely low activity. Cotransfection with subunit of NF-κB, c-rel in GK cells up-regulated CD9 promoter. Measuring endogenious CD9 expressions in response to poly(I:C) treatment in GK cells by RT-PCR showed no differences. Although the percoll purified white blood cells from head kidney and spleen of orange-spotted grouper showed no CD9 transcriptional difference after poly(I:C) treatment, IFNa and Mx expression showed significant upregulation.
In summary, the CD9 transcriptional regulation might associate with NF-κB, and might be inhibited by the infection of GIV. However, the transcription factors involving in CD9 promoter regulation during GIV infection remain further study.

並列關鍵字

Epinephelus coioides ； groupers ； GIV ； promoter ； CD9 ； luciferase assay ； poly(I:C) ； c-rel ； NF-κB

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