臺灣博碩士論文加值系統

在水產養殖中，亞硝酸鹽濃度過高造成生物體中毒，嚴重死亡是很常見的問題。有研究證實，高濃度的亞硝酸鹽會使魚體內的活性氧族群(Reactive oxygen species, ROS)上升，導致水生生物的死亡。也有研究證實錳超氧化物歧化酶可清除ROS保護細胞抵抗各種氧化壓力。為了提高水生生物抗亞硝酸耐受性，本研究利用細胞穿透肽TAT將錳超氧化物歧化酶送入魚鰭細胞(GF-1)內達到保護細胞對抗ROS所造成的損害。本研究建構石斑魚錳超氧化物歧化酶 (Manganese-dependent superoxide dismutase ,MnSOD)與含穿透肽的TAT-MnSOD基因，利用大腸桿菌BL21表現此兩種蛋白，接著以西方墨點法確定蛋白質表現，並以SDS-PAGE確定TAT-MnSOD的可溶性 (42± 1.2%)明顯高於MnSOD (0.01± 0.3%)，我們利用Nickel column純化蛋白質，並確認兩種蛋白皆保有SOD活性；將不同濃度的TAT-MnSOD、MnSOD處理GF-1細胞1小時後，以西方點墨法確認TAT-SOD較MnSOD有效率地進入細胞內。另外，我們發現GF-1細胞內的ROS會隨著NaNO2濃度的上升而上升，而細胞的活存率卻隨著NaNO2濃度的上升而下降。我們證實TAT-MnSOD能進入細胞內清除NaNO2所造成的ROS，並保護GF-1細胞對抗NaNO2所造成的傷害而提高細胞活存率。另外，我們將斑馬魚預先處理 TAT-MnSOD，進行亞硝酸鹽毒性測試，發現TAT-MnSOD處理較未經處理的組別在短時間內能提高斑馬魚的活存率。成功證實 TAT-MnSOD 能有效提升斑馬魚對抗亞硝酸鹽緊迫之活存率，未來若能有效應用於水產養殖業將對台灣水產養殖業有極大貢獻。

In aquaculture, nitrite concentration too high to cause serious death is a very common problem. It had been demonstrated that high concentrations of nitrite will increase active oxygen species (Reactive oxygen species, ROS) in fish and lead it to death. Studies have also confirmed that manganese superoxide dismutase (MnSOD) can remove ROS to protect cells against various oxidative stress. To enhance nitrite tolerance to aquatic organisms, we constructed cell penetrable MnSOD by fused cell penetrating peptide TAT and grouper MnSOD (TAT-SOD) to remove ROS induced by nitrite to protect cells. In this study, Manganese superoxide dismutase (MnSOD) and TAT-MnSOD fusion protein expression was exam by western blot and protein solubility of TAT-MnSOD (67 ± 3.1%) was significantly higher than that of MnSOD (11 ± 0.8%) by SDS-PAGE. Then we purified the protein by Nickel column and confirmed that both proteins had SOD activity. We demonstrated that TAT-MnSOD could efficiency transduce into GF-1 cells not MnSOD after protein treatment for 1hr by western blot. In addition, we found that ROS was increased with NaNO2 concentration in GF-1 cells, while the GF-1 cells survival rate was decreased with NaNO2 concentration. Finally, we demonstrated that TAT-MnSOD could entry cells and removed ROS induced by NaNO2 in cells to decrease the cell mortality caused by NaNO2. In addition, we demonstrated TAT-MnSOD protect zebrafish against the urgency of nitrite in vivo and TAT-MnSOD could effectively enhance the survival rate of aquatic organisms against nitrite toxic.

摘要 IV
Abstract VI
致謝 VIII
目錄 X
壹、 前言 1
材料與方法 10
一、 實驗材料 10
生物材料 10
藥品與儀器 10
二、 實驗方法 18
(一) 石斑魚RNA萃取及cDNA的製備 18
(二) 基因建構 19
(三)小量質體製備 21
(四)西方點墨法測試蛋白質表現 22
(五)蛋白質可溶性測試 23
(六)蛋白質純化 25
(七)酵素活性分析 26
(八) 重組融合蛋白質進入細胞的效能分析 27
(九) NaNO2影響GF-1細胞ROS的產生 28
(十) NaNO2 對GF-1細胞的毒性測試 29
(十一) 融合蛋白保護細胞測試 29
(十二)細胞活存率測試 30
(十三) NaNO2 對斑馬魚的毒性測試 31
(十四) TAT-MnSOD保護斑馬魚對抗NaNO2的毒性測試 31
参、結果 33
一、 pET22b-TAT-MnSOD及pET22b-MnSOD之 基因建構 33
二、 以西方點墨法測試TAT-MnSOD及MnSOD的蛋白質表現 33
三、 TAT-MnSOD及MnSOD之蛋白質可溶性 34
四、 TAT -MnSOD及MnSOD蛋白質之純化分析 35
五、 超氧化物岐化酶活性測試 35
六、 蛋白質進入細胞測試 35
七、 NaNO2對GF-1細胞內ROS的影響 36
八、 NaNO2對GF-1細胞的毒性測試及細胞型態變化 37
九、 分析TAT-MnSOD及MnSOD能降低細胞中因NaNO2誘發產生的ROS 37
十、 TAT-MnSOD及MnSOD保護GF-1細胞對抗NaNO2的毒性測試 38
十一、 NaNO2對斑馬魚的毒性測試 39
十二、 TAT-MnSOD保護斑馬魚對抗NaNO2的毒性測試 39
討論 41
伍、圖表 49
陸、參考文獻 63
柒、附錄 72

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