指導教授：許 輔臺灣大學：園藝學研究所林瑜珊Lin, Yu-ShanYu-ShanLin2014-11-292018-06-292014-11-292018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/263094金針菇免疫調節蛋白 FIP-fve 萃取自金針菇子實體，文獻指出其具有免疫調節、抗癌、抗過敏及減緩發炎反應等功效。二級結構分析顯示，FIP-fve 透過兩個單元體 N 端 α-helix 以疏水性鍵結形成雙元體。本實驗室前人研究指出 FIP-fve 不需經過抗原呈獻細胞修飾，而是透過與 MHC 及 TCR 分子鍵結活化 T 細胞，因此推測FIP-fve 作用方式類似超抗原。本研究目的有二，一為證實雙元體結構對於 FIP-fve 的免疫調節活性是必要的，二為找出 FIP-fve 與 MHC 和 TCR 具交互作用之證據。為探討雙元體結構對 FIP-fve 免疫調節活性之影響，將 1-13 胺基酸缺失或全長 FIP-fve cDNA構築於 pET-32a(+) 載體，並透過大腸桿菌系統表現得分子量皆約 26 kDa 之 His-FIP-fve 1-114 和 His-FIP-fve 14-114 蛋白。以 enterokinase 切除 His-tag 後，經質譜比對確認成功取得 rFIP-fve 1-114 以及 rFIP-fve 14-114。免疫調節活性試驗方面，片段缺失 rFIP-fve 14-114 刺激小鼠脾臟細胞分泌 IFN-γ 能力較 rFIP-fve 1-114 弱，且無法刺激小鼠脾臟細胞 IFN-γ 及 IL-2 基因表現。綜合以上結果，推測失去雙元體結構造成 FIP-fve 免疫調節活性降低。在 FIP-fve 類超抗原性質探討上，透過共同免疫沉澱法可知 FIP-fve 與表面分子 MHC class II 具交互作用，另外發現 FIP-fve 可被 MHC class I 及其 Isotype IgG2a 抗體捕捉。然而，本研究尚未透過共同免疫沉澱法看見 FIP-fve 與 TCR 連結之證據，因此僅能證實 FIP-fve 能連結 APCs 表面分子 MHC class II。FIP-fve, an immunomodulatory protein isolated from Flammulina velutipes, has been demonstrated to have activities of immunomodulatory, anti-cancer, anti-allergic and suppression of inflammation. Based on a secondary structure analysis, we known that two FIP-fve monomers form homodimer through the hydrophobic interaction between N-terminal α-helics. Previous studies suggested that FIP-fve can mediate T cells activation through the linkage between T cell receptors (TCR) on T cells and MHC molecules on antigen present cells (APCs) without processing procedure of APCs. This process is similar to the mechanism of superantigen-mediated immune response. The aim of this study was to demonstrate the necessity of dimer structure for FIP-fve immunomodulatory activity and to prove the directly binding of FIP-fve with MHC and TCR. To study the relationship between the dimer structure and immunomodulatory activity of FIP-fve, we constructed the full length 1-114 FIP-fve cDNA and truncated 14-114 FIP-fve cDNA on pET-32a(+) vector and expressed the His-fusion protein His-FIP-fve 1-114 and His-FIP-fve 14-114. After digested by enterokinase to remove the His-tag residue, rFIP-fve 1-114 and rFIP-fve 14-114 were purified with FPLC system. The ability of rFIP-fve 14-114 to stimulate IFN-γ production on murine splenocytes was lower than rFIP-fve 1-114. Incubating murine splenocytes with rFIP-fve 14-114 could not elevate the gene expression of IFN-γ and IL-2. According to the above results, we suggested that the dimerization might be critical for FIP-fve immunomodulatory activity. On the other hand, FIP-fve and RAW 264.7 cell lysate were co-immunoprecipitated to determine superatigen-like characterization of FIP-fve. Results indicated that FIP-fve could interact with MHC class II molecule directly. Interestingly, we also found that FIP-fve was pulled down by both anti-MHC class I antibody and IgG2a which was isotype of anti-MHC class I antibody. However, no co-immunoprecipitated evidence showed that FIP-fve bound with TCR in this study. In conclusion, we proved that FIP-fve directly bind with MHC class II molecule.口試委員會審定書 I 致謝 II 摘要 IV Abstract V 目錄 VII 圖目錄 IX 縮寫表 X 第一章 前人研究 1 第一節　金針菇簡介 1 第二節　其他金針菇成分與功效介紹 2 第三節　金針菇免疫調節蛋白 FIP-fve 簡介 5 第四節　超抗原簡介 10 第二章　研究動機與目的 12 第三章　材料與方法 13 第一節　金針菇免疫調節蛋白 FIP-fve 純化 14 第二節　變性膠體電泳分析 (SDS-PAGE) 17 第三節　蛋白質濕式轉印 19 第四節　西方轉漬法 (western blotting) 20 第五節　重組蛋白 rFIP-fve 1-114 及truncated rFIP-fve 14-114 基因選殖與構築 21 第六節　重組蛋白 rFIP-fve 1-114 及truncated rFIP-fve 14-114 表現與純化 25 第七節　膠體內水解 (In gel digestion) 27 第八節　小鼠脾臟細胞取得 28 第九節　酵素連結免疫吸附分析 (Enzyme-linked immunosorbent assay, ELISA) 30 第十節　細胞總 RNA 抽取與反轉錄 31 第十一節　即時定量聚合酶鏈鎖反應 (Real-time PCR) 32 第十二節　巨噬細胞株 RAW 264.7 細胞培養 34 第十三節　共同免疫沉澱法 (Co-immunoprecipitation) 35 第十四節　統計分析 38 第四章 研究結果 39 第一節　重組蛋白 rFIP-fve 1-114 及 truncated rFIP-fve 14-114 選殖與純化 39 第二節　重組蛋白 rFIP-fve 1-114 及 truncated rFIP-fve 14-114 於小鼠脾臟細胞之免疫活性 42 第三節　以共同免疫沉澱法探討 FIP-fve 之類超抗原特性 43 第五章　討論 46 第一節　重組蛋白 rFIP-fve 1-114 及 truncated rFIP-fve 14-114 蛋白純化探討 46 第二節　片段缺失 truncated rFIP-fve 14-114 蛋白功能探討 47 第三節　FIP- fve 與表面分子親和性之探討 48 第六章　結論與未來展望 51 參考文獻 524501145 bytesapplication/pdf論文公開時間：2017/08/25論文使用權限：同意無償授權超抗原His 融合蛋白同型雙元體免疫調節蛋白共同免疫沉澱[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263094/1/ntu-103-R01628205-1.pdf