銀耳TFP1蛋白對小鼠腹腔巨噬細胞免疫調節作用及異體表現之研究

Investigation of the Immunomodulatory Effects of the TFP1 Protein from Tremella Fuciformis Berk. on Mouse Peritoneal Marcophages and its Heterologus Expression

洪志良(Chih-Liang Hung)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01581

摘要

本研究以銀耳 (Tremella fuciformis Berk) 免疫調節蛋白 TFP1 為材料，目的在深入探討 TFP1 之生化特性，以及研究 TFP1 對小鼠腹腔巨噬細胞的免疫調節活性與活化路徑，並建立酵母菌的異體表現系統。在探討 TFP1 的生化特性方面，根據質譜儀分析之結果，發現 native TFP1 之分子量約 11.0 kDa，與其基因轉錄後預期的分子量 (11.4 kDa) 相近，為重組 TFP1 經膠體層析與SDS-PAGE 電泳所得值 (約 24.0 kDa) 之半，因此推測 TFP1 在自然環境下可能為同質雙元體。在研究 TFP1 調節免疫的作用方面，實驗發現 TFP1 能夠活化小鼠腹腔巨噬細胞產生 TNF- a 及 IL-1b，同時也能提高 TNF-a, IL-1b, IL-6, IL-12p35, IL-12p40, CCL3, 以及 CXCL10 的 mRNA 表現，同時流式細胞分析發現 TFP1 可增加腹腔巨噬細胞吞噬能力及CD86 之表現，且電泳位移分析 (EMSA assay) 顯示 TFP1 能夠活化 NF-kB 轉錄因子；另一方面，TFP1 活化小鼠腹腔巨噬細胞的作用，會受  anti-TLR2/TLR4 抗體中和處理以及 TLR4-/- 基因缺陷所抑制，故推測 TFP1 活化腹腔巨噬細胞的作用與 TLR2/TLR4 活化 NF-kB 之路徑高度相關。另一方面，將 TFP1 基因轉殖置入 pYEX-S1 載體，於 Saccharomyces cerevisiae 酵母菌株 DY150 表現得到重組 TFP1及帶有 His-tag 的 TFP1。本研究之結果進一步釐清 TFP1 活化巨噬細胞的機轉，並有助於將 TFP1 開發成飼料或調節免疫產品。

關鍵字

銀耳 ； 免疫調節蛋白 ； 腹腔巨噬細胞 ； 酵母菌 ； 異體表現

並列摘要

TFP1 is an immunomudulatory protein previously purified from jelly fungi (Tremella fuciformis Berk).  The objectives of this study was to clarify the biochemical characteristics of TFP1, to further investigate its regulating function and its activation pathway on mouse peritoneal macrophages  as well as to construct its yeast expression system.  Mass spectrometry analyses obtained the molecular weight of native TFP1 (11.0 kDa), agreeing its genetically translated value (11.3 kDa).  Based on gel-filtration and SDS-PAGE analyses, the recombinant TFP1 showed a molecular mass of 24 kDa, suggesting that TFP1 could be a homodimer protein.  In addition, TFP1 activated mouse peritoneal macrophages, increased the production of TNF-a and IL-1b and enhanced the mRNA expression of TNF-a, IL-1b, IL-6, IL-12p35, IL-12p40, CCL3, and CXCL10.  Flow cytometry analysis demonstrated that TFP1 increased the phagocytosis activity and CD86 expression by the cells.  Moreover, EMSA result showed a TFP1-induced activation of the transcription factor NF-kB.  The stimulatory effects of TFP1-promoted TNF-a secretion on macrophages were inhibited under antibody neutralization and TLR4 deficiency (TLR4-/-).  Based on these results, TLR2/TLR4 signaling pathway was suggested to be involved in TFP1-induced NF-kB activation.  Furthermore, TFP1 gene was cloned into pYEX-S1 vector and expressed by Saccharomyces cerevisiae to produce recombinant TFP1 and His-tagged TFP1.  This study clarified the mechanism and activities of TFP1 on mouse peritoneal macrophages and also provided more information on the application of feed and food utilization.

並列關鍵字

Tremella fuciformis Berk ； immunomudulatory protein ； peritoneal macrophages ； yeast ； heterologus expression

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