裂褶菌21kDa免疫激活蛋白純化及其 細胞毒殺能力之影響

Abstract

近年來許多報告指出，菇蕈萃取物當中的蛋白質，具有活化體內多種免疫細胞、促進免疫細胞的細胞分裂、提高巨噬細胞的吞噬能力與自然殺手細胞的活性、刺激免疫細胞分泌細胞激素的能力。 本研究從裂褶菌的培養液當中，純化出裂褶菌所分泌出具有免疫激活功能的蛋白。所純化出的蛋白質經由許多不同的特性分析，了解其所具有的生理特性，以及激活蛋白刺激的免疫機能。發現所純化出的免疫激活蛋白經由SDS-PAGE結果顯示，所純化的蛋白質為一單條帶，LC/MS/MS的分析約為21KDa，在醣蛋白染色的結果中發現，無醣蛋白的反應；血液凝集實驗發現並不會造成血液凝集的效能，與真菌免疫調節蛋白相比，真菌免疫調節蛋白雖不具有醣基，確會誘發血液凝集的效果因此推論本實驗所純化的免疫激活蛋白不屬於真菌免疫調節蛋白的家族。 免疫激活功能實驗中發現，以不同濃度的激活蛋白刺激脾細胞的增生，在蛋白濃度27 (μg/mL)的影響下脾細胞與控制組相比有顯著差異(p<0.05)，在誘發小鼠自然殺手細胞活性中也發現，蛋白濃度27 (μg/mL)刺激的處理組上，攻擊YAC-1的能力最為明顯而且與控制組相比也有顯著的差異(p<0.05)，但在進行誘發IFN-γ產生的實驗中卻沒有顯著的誘發效果(p>0.05)。總結以上，本研究所純化出的蛋白質可以刺激脾細胞分裂，誘發自然殺手細胞的活性，可做為一種免疫激活物增強免疫機能。

Recently studies demonstrated that purified protein from mushroom could activate immune cell, stimulate proliferation of immune cell, advance phagocytosis of macrophage, and enhance ability of natural killer cell (NK cell). In this study, immunostimulant protein from Schicophyllum commune was purified by 80% saturation of ammonia sulfate, DEAE ion exchange and Sepharex 75 gel filtration. SDS-PAGE analysis showed that the protein has a molecular weight of 21KDa and periodic acid Schiff reagent test revealed that the purified protein was not glycoprotein. Immunostimulant protein could not agglutinate ICR misce red blood cells, but stimulated the proliferation of mouse splenocyte with variant protein concentration, especially in the treatment of 27 μg/mL protein was significant increased (p<0.05). IFN-γ which mainly secreted by T cell and NK cell activate B cell, cytotoxic T cell, and macrophage. This study showed that purified protein did not affect the IFN-γ secretion of ICR mice. In cellular cytotoxity experiment, derived monocyte from ICR mouse splenocyte cocultured with immunostimulant protein. The result indicated that immunostimulant protein enhanced cytotoxic ability of NK cell and the treatment concentration with 27 μg/mL was the most significant higher than control (P<0.05). This study clearly demonstrated that the purified protein activated the proliferation of splenocyte and cytotoxic ability of NK cell, is an immune stimulus could be helpful to strength the immunity.