蛹虫草免疫调节蛋白分离纯化、结构表征与免疫调节活性研究

Isolation, purification, structural characterization and immunomodulatory activity of immune regulatory proteins from Cordyceps militaris

中文关键词:  蛹虫草  蛋白质  纯化  结构表征  巨噬细胞  免疫调节作用

英文关键词:Cordyceps militaris  protein  purification  structural characterization  macrophages  immunomodulatory activity

中文摘要:

      目的 探究蛹虫草蛋白质的免疫调节活性和一级结构。方法 通过碱溶酸沉法提取蛹虫草蛋白质, 采用阴离子交换层析法和葡聚糖凝胶层析法对其进行分离纯化, 通过建立小鼠巨噬细胞免疫调节模型对各组分进行活性评价, 得到免疫活性最佳的组分, 通过基质辅助激光解吸飞行时间质谱法测得其分子量大小, 通过蛋白质谱解析出其蛋白质一级结构。以脂多糖(lipopolysaccharide, LPS)作为阳性对照, 通过小鼠巨噬细胞研究其对巨噬细胞RAW264.7增殖活性、吞噬率、一氧化氮(nitrogen monoxide, NO)分泌量及免疫因子白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-6 (interleukin-6, IL-6)、干扰素-γ (interferon-γ, IFN-γ)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)释放量等的影响。结果 分离纯化出的蛹虫草免疫调节蛋白CMP 2b由196个氨基酸组成, 分子量为22.460 kDa; CMP 2b可显著提高巨噬细胞的增殖能力和吞噬能力, 细胞增殖率最高达到了空白对照组的(152.91±5.55)%, 吞噬能力提高了23.34%; CMP 2b可促进巨噬细胞分泌NO、活性氧(reactive oxygen species, ROS)、IL-1β、IL-6、IFN-γ及TNF-α, 其中NO分泌水平达到了空白对照组的2.31倍, ROS分泌水平达到阳性对照组LPS的82.33%, TNF-α、IL-1β、IFN-γ和IL-6分泌量分别为空白对照组的5.51、17.42、4.18和26.75倍, 显著增强了巨噬细胞免疫调节能力。结论 作为近年来兴起的一种优质珍稀食用菌, 蛹虫草蛋白质在体外研究中显示出较强的免疫增强作用, 可为蛹虫草作为免疫调节剂的开发提供新思路。

英文摘要:

      Objective To explore the immunomodulatory activity and primary structure of Cordyceps militaris protein. Methods Cordyceps militaris protein was extracted by alkali-soluble acid precipitation method, and separated and purified by anion exchange chromatography and glucan gel chromatography. The activity of each component was evaluated and the best immunoactive component was obtained by establishing mouse macrophage immune regulation model. Its molecular weight was measured by matrix-assisted laser desorption time-of-flight mass spectrometry and its primary protein structure was determined by protein spectroscopy. With the lipopolysaccharide (LPS) as a positive control, the proliferative activity, phagocytosis rate, nitrogen monoxide (NO) secretion and the release of immune factor interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) of RAW264.7 cells in mouse macrophages were studied. Results The isolated and purified immunomodulatory protein CMP 2b was composed of 196 amino acids and its molecular weight was 22.460 kDa. The CMP 2b could significantly improve the proliferation ability and phagocytosis ability of macrophages, the cell proliferation rate reached the highest (152.91±5.55)% of blank control group, phagocytosis ability increased by 23.34%. In addition, CMP 2b could promote the secretion of NO, reactive oxygen species (ROS), IL-1β, IL-6, IFN-γ and TNF-α by macrophages, in which the secretion of NO was 2.31 times of that of blank control group, and the secretion of ROS was 82.33% of LPS of positive control group. The secretion levels of TNF-α, IL-1β, IFN-γ and IL-6 were 5.51, 17.42, 4.18 and 26.75 times of the blank control group, respectively, which significantly enhanced the immune regulation ability of macrophages. Conclusion As a high-quality rare edible fungus emerging in recent years, the protein of Cordyceps militaris has strong immune enhancing effect in vitro, and the results of the study may provide new ideas for the development of Cordyceps militaris as an immunomodulator.