裂褶菌30 kDa免疫細胞活化蛋白之純化及其抗腫瘤活性之研究

Abstract

本研究從裂褶菌 (Schizophyllum commune) 發酵液中分離純化出免疫激活及抗腫瘤增殖功能之蛋白質BCⅡ。裂褶菌發酵液經硫酸銨沉澱、透析復溶後，再利用快速蛋白質液相層析系統 (FPLC) 以HIC疏水性層析、Mono-Q陰離子交換層析和Superdex 75膠體過濾層析後得到純化蛋白質BCⅡ。以SDS-PAGE蛋白質電泳和膠體過濾分析後，推估BCⅡ為一單體，分子量約為30 kDa，且由periodic acid / Schiff reagents染色後發現BCⅡ不為醣蛋白。藉由MTT assay發現到BCⅡ在濃度50、25 μg/ml時，具抑制小鼠肺癌細胞 (LLC)增殖作用；並且BCⅡ對小鼠脾細胞無細胞毒性。在免疫激活試驗方面，濃度5 μg/ml BCⅡ可直接促進自然殺手細胞 (NK cell) 之毒殺活性；不同濃度之BCⅡ亦可刺激巨噬細胞RAW 264.7產生一氧化氮 (nitric oxide) 和TNF-α。一氧化氮之產生有劑量依賴性的現象 (dose-dependent)；TNF-α則是於25 μg/ml BCⅡ時，有最高的產生量。綜合本實驗之結果，裂褶菌蛋白BCⅡ具有免疫激活之特性，並具有抗腫瘤增殖之活性，有潛力在臨床藥學上發展及應用。

From the fermentation liquid of the mushroom Schizophyllum commune, a monomer protein, BCⅡ, with immunostimulative and antitumor activities had been isolated. The purification procedure was involved (NH4)2SO4 precipitation, hydrophobic interaction chromatography on Phenyl sepharose, ion exchange chromatography on Mono-Q and gel filtration on Superdex 75 by fast protein liquid chromatography (FPLC). The molecular mass of BCⅡ was 30 kDa as estimated via SDS-PAGE and gel filtration. Further staining using the periodic acid / Schiff reagents showed that BCⅡ was not a glycoprotein. BCⅡshowed antiproliferative activity toward mouse Lewis lung carcinoma (LLC) at BCⅡ concentration of 50, 25 μg/ml, respectively. And showed no cytotoxicity to mouse spleen cells by MTT assay. The immunostumulative activities of BCⅡ was also demonstrated in this study, which activate directly cytotoxicity of nature killer (NK) cell and macrophage RAW 264.7 to produce nitric oxide and TNF-α. BCⅡ stimulated macrophage RAW 264.7 to produce nitric oxide in a dose-dependent effect. The highest level of TNF-α was exhibited at BCⅡ concentration of 25μg/ml. Based on the results obtained in the present study, BCⅡ is a potent immune stimulant and could develop potentially using in medicine field.