白木耳中免疫調節蛋白活性分析與基因選殖之研究

Functional Analysis and Molecular Cloning of Immunomodulatory Protein from Tremella fuciformis

張安如(An-Ju Chang)

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(2006年)

https://doi.org/10.6342/NTU.2006.00998

摘要

白木耳 (Tremella fuciformis Berk) 為重要食藥用菇類之一，本研究以白木耳乾燥子實體為材料，經硫酸銨沈澱及 DE-52 陰離子交換管柱純化出具有免疫調節活性的蛋白區分物 TFPs，進一步以 HiTrap Q 陰離子交換層析與膠體過濾層析將 TFPs 分離出兩個蛋白，分別命名為TFP1 與 TFP2，其分子量分別約為 24 kDa與 40 kDa，以等電聚焦電泳分析發現兩者之 pI 值皆為 3.45。TFP1 和 TFP2 經過醣蛋白染色試驗後，發現兩者皆非醣蛋白，經過血球凝集試驗後，發現兩者皆不具有凝集小鼠紅血球之活性。經過體外免疫活性試驗發現白木耳蛋白 TFP1 與TFP2 均可直接活化 RAW264.7 巨噬細胞分泌 nitric oxide 與 TNF-α (p ＜ 0.05)，此外，TFP1 與 TFP2 也能夠刺激小鼠脾臟細胞增生，而TFP1蛋白在100℃ 或 -80℃ 處理後皆能保持其刺激巨噬細胞分泌 NO 的免疫活性。此外，以細胞融合技術建立 3 株能夠辨識白木耳蛋白 TFPs 之單株抗體，有利於日後之純化與檢測，以及進行相關的蛋白質體學之研究。另一方面以白木耳菌絲體為原料，根據 TFP1 蛋白 N 端胺基酸序列合成 TFP1 之 degenerate primers，嘗試以3’ 及5’ rapid amplification cDNA ends (RACE) 的方法針對 TFP1 進行 cDNA 選殖，但未取得符合序列之基因；由二維電泳與西方轉漬分析發現用來做為原料基因選殖的菌絲體可能沒有表現目標蛋白或表現量很低，遂使得選殖工作遭到困境。序列比對分析各選殖片段產物後發現，白木耳菌絲體的基因可能與 Cryptococcus neoformans var. neoformans JEC21的基因有高度相關性。綜合本研究結果得知，TFP1 與 TFP2 是白木耳中重要的免疫調節活性物質，可提升宿主相關免疫反應，極具醫藥以及臨床發展之潛力。

關鍵字

白木耳 ； 免疫調節蛋白 ； 基因選殖

並列摘要

Tremella fuciformis Berk is not only an edible mushroom but also known to possess pharmacological properties. The new immunomodulatory protein TFPs were purified from the dried fruiting body of T. fuciformis by ammonium sulfate precipitation and DE-52 anion-exchange chromatography. Proteins from the peak fractions were collected and further purified using HiTrap Q ion-exchange and gel filtration chromatography. Finally, two distinct proteins, TFP1 and TFP2 were obtained. SDS-PAGE analysis showed that the molecular weights of TPF1 and TFP2 were 24 kDa and 40 kDa, respectively. Isoelectric focusing electrophoresis revealed that the pI values of both proteins were both approximately 3.45. Both TFP1 and TFP2 were not glycoproteins and none of them could agglutinate mouse red blood cells. In addition, TFP1 and TFP2 could directly activate RAW264.7 macrophages and enhance the production of both nitric oxide (NO) and tumor necrosis factor-alpha (p＜0.05). TFP1 and TFP2 were also capable to stimulate the proliferation of murine splenocytes in vitro. TFP1 remained fairly good activities on stimulating the secretion of NO by RAW264.7 macrophages after different temperature treatments such as 100℃ and -80℃. Moreover, three monoclonal antibodies which recognize TFPs specifically were established, and they could be utilized for purification and other proteomic studies in the future. Furthermore, cDNA cloning of TFP1 protein from the mycelium of T. fuciformis was also performed. TFP1 degenerate primers based on the N-terminal amino acid sequence were made, and 3’ rapid amplification of cDNA ends (RACE) and 5’ RACE PCR were carried out in order to clone the full-length TFP1 cDNA. However, no sequence matched TFP1 was obtained. Results from two-dimensional electrophoresis and Western blotting analysis suggested that TFP1 gene might be expressed at a low level or even not expressed in the mycelium of T. fuciformis, which might contribute to the failure of gene cloning. BLASTX sequence analysis showed that there was a high correlation between T. fuciformis and Cryptococcus neoformans var. neoformans JEC21 genes. This study suggested that TFP1 and TFP2 were important immunomodulatory components in T. fuciformis and could strengthen the immunity of the host.

並列關鍵字

Tremella fuciformis ； immunomodulatory protein ； gene cloning

參考文獻

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