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生物活性蛋白是药用真菌中一类重要的活性功能因子.云芝是中国传统的药用真菌,具有抗病毒、抗肿瘤和免疫调节等活性.通过硫酸铵沉淀、阴离子交换层析和凝胶过滤层析等过程,从云芝子实体中分离纯化得到一种新的免疫调节蛋白(命名为TVC).TVC在SDS-PAGE结果中呈现分子量为15kD的单一条带,而在Native-PAGE结果中表现分子量为30kD,表明天然的TVC为同源二聚体.等电聚焦电泳表明,TVC的等电点pI为4.0,为酸性蛋白质,根据periodic acid/Schiff试剂染色反应表明,TVC不含糖,且对大鼠红细胞没有凝集作用,表明TVC缺乏凝集素的典型特征.免疫活性测定,应用流式细胞仪检测发现TVC能增强小鼠脾淋巴细胞的增殖,但对CD4+,CD8+等T细胞无影响;TVC以剂量依赖的方式对人外周血淋巴细胞有增殖活性,同时与刀豆蛋白A有联合增殖效应;TVC还能以剂量依赖方式提高脂多糖LPS诱导的小鼠巨噬细胞RAW264.7产生NO和肿瘤坏死因子TNF-α的产量,表明TVC是一种免疫刺激剂,能够提高机体的免疫反应.N端测序和蛋白质MALDI-TOF/TOF质谱,没有在蛋白质数据库中发现同源蛋白质,说明TVC是一个新的免疫调节蛋白.
Biologically active protein is an important active functional factor in medicinal fungi.Close is a traditional Chinese medicinal fungus with anti-virus, anti-tumor and immunomodulatory activity.After ammonium sulfate precipitation, anion exchange chromatography and gel (TCA), a new immunomodulatory protein (named as TVC) was isolated and purified from the polysaccharides of E. fortunei.TTVC showed a single band with molecular weight of 15 kD in SDS-PAGE, Showed a molecular weight of 30 kD, indicating that the native TVC is a homodimer.Isalytic electrophoresis showed that the isoelectric point pi of TVC was 4.0, which was acidic protein, indicating that TVC was free of sugars according to the periodic acid / Schiff reagent staining reaction , And no agglutination of rat erythrocytes, indicating that TVC lacks the typical features of lectin.Immunofactivity assay, using flow cytometry showed that TVC can enhance the proliferation of mouse splenic lymphocytes, but the CD4 +, CD8 + T cells such as no TVC can proliferate human peripheral blood lymphocytes in a dose-dependent manner and have a combined proliferative effect with concanavalin A. TVC can also increase lipopolysaccharide LPS-induced mouse macrophage RAW production in a dose-dependent manner 264.7 production of NO and tumor necrosis factor TNF-α production, indicating that TVC is an immune stimulator, can improve the body’s immune response.N-terminal sequencing and protein MALDI-TOF / TOF mass spectrometry, no homologous protein was found in the protein database , Indicating that TVC is a new immunomodulatory protein.