臺灣博碩士論文加值系統

文獻指出金針菇免疫調節蛋白質 FIP-fve 以非共價鍵結的形式，藉由兩個單元體 N 端 alpha-helix 的疏水性作用形成雙元體，並進一步由 beta-sheet 穩定雙元體結構而以雙元體形式存在於自然界；本研究室之前亦提出 fve 的作用機制類似超抗原的假說，必須與 MHC 分子及 TCR 鍵結才能活化 T 細胞。本研究目的在證明，雙元體構形是 fve 免疫調節活性之必要條件。本研究的架構分成兩部份，第一部份純化缺失不同胺基酸序列之 truncated fve 並探討 fve 雙元體與免疫調節活性之關係，第二部份則分析 fve 保存期限以及建立 fve 免疫分析平台。
為了確認 FIP-fve 雙元體構形與免疫調節活性之關係，使用大腸桿菌表現系統生產七種缺失部分片段的 fve 蛋白質，其中僅 truncated fve-28-103 成功表現及純化，truncated fve-28-103 因為胺基酸序列缺失而無法形成雙元體，將 truncated fve-28-103 加以螢光標定，觀察其與巨噬細胞及 T 細胞結合情形，並與脾臟細胞共同培養，測定培養液中 IFN-gamma 含量，以確認活性。結果顯示 truncated fve-28-103 與巨噬細胞的結合能力較 native fve 低，且不與 T 細胞結合，同時失去活化脾臟細胞產生 IFN-gamma 之能力。由結果可推測，fve 無法形成雙元體即不具免疫調節活性。
為了評估食品中所添加 fve 的保存期限，將 native fve 使用不同殺菌條件處理並經過不同保存時間後進行電泳分析；以中性環境 121℃ 處理20分鐘或以 138℃ 處理18秒，fve 至少能夠在 25℃ 保存半年，而以 130℃ 處理12秒或以 121℃ 處理20分鐘則至少可在 4℃ 保存半年。另外，為了有效檢測未來開發之 fve 保健食品中 fve 的含量，本研究製作 fve 單株抗體建立 fve 的 sandwich ELISA，得到其濃度標準曲線 (y = 0.3464x – 0.8264) 之線性係數 r2 = 0.9403，定量 fve 濃度的範圍為103—106 pg/mL。由以上結果可知，fve 經處理後之保存期限至少半年，而 fve 的免疫分析平台則可應用於檢測食品中 fve 含量。

FIP-fve, a fungal immunomodulatory protein isolated from Flammulina Velutipes, has been reported to be a dimer in its native form. The main driving force of fve dimerization was the hydrophobic interactions between the N-terminal alpha-helices of the two monomers. The fve dimer was further stabilized by the beta-sheet which consisted of the N-terminal beta-strands of the two monomers. Previous study also suggested that fve was a superantigen-like immunomodulatory protein, which activated T cells through the binding of both MHC molecules and the T cell receptor. Our objective in this study was to prove that the dimerization was critical and necessary for the immunomodulatory activity of fve. This study included two parts: (1) to study the relationship between fve dimer and its immunomodulatory activity and (2) to investigate the expiration date of native fve. We also established a sandwich enzyme-linked immunosorbent assay (ELISA) to determine the concentration of fve.
To study the relationship between fve dimer and its immunomodulatory activity, we used E.coli expression system to produce seven truncated fve. However, only truncated fve-28-103, which could not form dimer due to the deletion of 1- 27 residues at the N-terminus, was successfully expressed and purified. The fve-28-103 was then labeled with FITC to confirm its binding activity with peritoneal macrophages and T cells. We also detected the IFN-gamma production by murine splenocytes which were induced by truncated fve-28-103. Our results showed that the binding ability of truncated fve-28-103 was lower than that of native fve toward peritoneal macrophages. The binding was not significant on T cells. Moreover, truncated fve-28-103 did not activate splenocytes to produce IFN-gamma. According to these results, we concluded that the dimerization was essential for the activity of fve.
We have evaluated the expiration date of native fve in food under different sterilization processes and storage conditions. The SDS-PAGE analysis showed that little degradation of fve was observed after the treatment with 121℃ for 20 minutes or 138℃ for 18 seconds. Then, the fve could be stored at 25℃ at least for 6 months. Fve treated with 121℃ for 20 minutes or 130℃ for 12 seconds could also be stored at 4℃ at least for 6 months. To develop an analytical method for fve, we have further established a specific and sensitive sandwich ELISA for fve quantification using anti-fve monoclonal antibodies. The linear quantitative concentration ranged from 103-106 pg/mL with a multiple coefficient of determination r2=0.9403. As a result, fve could be stored at least for 6 months under different treatments. This fve ELISA method could be applied for the determination of fve content in food samples.

誌謝.....................................................I
摘要.....................................................II
Abstract................................................III
目錄.....................................................V
表目錄....................................................VII
圖目錄....................................................VII
壹、 前人研究..........................................1
一、 金針菇簡介.........................................1
二、 金針菇免疫調節蛋白質 FIP-fve 的前期研究................1
三、 超抗原之介紹........................................5
四、 單株抗體原理........................................6
五、 研究動機與目的.......................................6
貳、 材料...............................................8
一、 實驗動物............................................8
二、 藥品及試劑套組.......................................8
三、 器材..............................................10
參、 方法..............................................12
一、 金針菇免疫調節蛋白質 FIP-fve 製備.....................12
(一) 純化 FIP-fve......................................12
(二) 蛋白質濃度測定......................................13
(三) 變性膠體電泳分析 (sodium dodecyl sulfate polyacrylamide gel electrophoresis).....................................14
二、 表現與純化 GST-fve.................................15
(一) 小量表達 GST-fve...................................15
(二) 大量表達 GST-fve...................................16
(三) 純化 GST-fve......................................16
(四) 切除 GSTtag.......................................17
(五) 純化 truncated fve................................17
1. 使用 GSTrap FF 管柱及 HiTrap Benzamidine FF 管柱....17
2. 使用 HiTrap Q 管柱.................................17
(六) 蛋白質濕式轉印......................................18
(七) 西方轉漬分析 (western blotting).....................18
三、 truncated fve 之細胞試驗...........................19
(一) 取得小鼠脾臟細胞....................................19
(二) 測定細胞激素 IFN-g .................................20
(三) 純化小鼠 T 淋巴細胞.................................21
(四) 取得小鼠腹腔巨噬細胞.................................21
(五) truncated fve 與小鼠 T 淋巴細胞及腹腔巨噬細胞之結合關係 .22
1. 製備螢光標定蛋白質：.................................22
2. truncated fve 與 T 淋巴細胞的結合關係：...............22
3. truncated fve 與腹腔巨噬細胞的結合關係：..............23
四、 金針菇免疫調節蛋白質之單株抗體製作......................24
(一) 製作融合瘤細胞株....................................24
(二) 生產並純化 anti-fve 單株抗體.........................26
(三) 抗體效價測試........................................27
(四) 抗體專一性測試......................................28
五、 建立 native fve 的 ELISA 免疫分析平臺................29
(一) fve 抗體生物素化 (biotinylation)...................29
(二) 利用酵素連結免疫分析法分析生物素化的 fve 抗體............29
(三) fve 捕捉及偵測抗體配對之 ELISA 分析...................29
(四) 建立 native fve 濃度標準曲線.........................30
六、 native fve 保存試驗................................31
(一) 樣品處理...........................................31
(二) 變性膠體電泳分析 (sodium dodecyl sulfate polyacrylamide gel electrophoresis).....................................31
七、 統計分析...........................................32
肆、 結果..............................................33
一、 表現與純化融合蛋白質GST-fve..........................33
二、 native fve 及 truncated fve 之細胞試驗..............34
三、 native fve 保存試驗................................35
四、 native fve 單株抗體製作.............................36
五、 建立 ELISA 免疫分析平台.............................36
六、 建立 fve 濃度標準曲線...............................37
伍、 討論..............................................38
一、 truncated fve 製備流程探討 .........................38
二、 truncated fve 28-103 功能分析......................39
三、 native fve 保存試驗................................40
四、 native fve 捕捉及偵測抗體配對 ELISA 分析..............41
五、 建立 native fve 濃度標準曲線.........................41
陸、 結論及未來展望......................................43
參考文獻 .................................................44

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