摘要
为研究食用菌多肽的免疫调节作用,以杏鲍菇干粉为原料,通过碱提酸沉法提取杏鲍菇粗蛋白,再经胃蛋白酶、胰蛋白酶复合酶解,将获得的杏鲍菇粗蛋白酶解液通过超滤分离、Sephadex G-15葡聚糖凝胶柱层析及高效液相色谱法分离进行纯化,并构建RAW264.7巨噬细胞模型,测定其增殖率、NO释放率、吞噬率和细胞因子mRNA表达量,对各组分进行筛选。结果表明,>10、5~10、3~5 kDa和<3 kDa 4种分子质量不同的超滤纯化组分中,<3 kDa组分的免疫活性最高。Sephadex G-15和高效液相色谱法分离获得免疫活性最高的组分Peak 2-2。利用液相色谱-串联质谱对Peak 2-2组分进行de novo测序,根据可信度评分(≥99),筛选获得51条多肽序列。最后,利用分子对接手段确定杏鲍菇多肽DFPALR和LLGVD与TLR4蛋白的结合稳定,通过分子动力学模拟技术确定两条多肽与TLR4蛋白的结合是可自发的、稳定的。本研究表明杏鲍菇多肽具有良好的免疫调节活性。
In order to study the immunomodulatory effects of peptides derived from edible mushroom,crude protein was extracted from dried powder of Pleurotus eryngii and sequentially hydrolyzed with pepsin and trypsin.The resulting hydrolysate was separated and purified by ultrafiltration with membranes with different molecular mass cutoffs(MMCOs of 3,5 or 10 kDa),Sephadex G-15 column chromatography and high performance liquid chromatography(HPLC).The effects of the products obtained at the end of each step on the proliferation,NO release,phagocytic activity,and mRNA expression of inflammatory cytokines and inducible nitric oxide synthase(iNOS)in RAW264.7 macrophages were assessed.The results showed that among all ultrafiltration fractions,the one with a molecular mass of less than 3 kDa exhibited the highest immunoregulatory activity.After purification by Sephadex G-15 column chromatography and HPLC,fraction Peak 2-2 was obtained,with the highest immunomodulatory activity.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used for de novo sequencing of Peak 2-2,resulting in the identification of 51 peptide sequences with a confidence score greater than 99.Finally,molecular docking results confirmed the stable binding of P.eryngii peptides DFPALR and LLGVD to toll-like receptor 4(TLR4),and molecular dynamics simulation showed that the binding was spontaneous and stable.This study indicated that P.eryngii peptides possessed significant immunomodulatory activity.
作者
田江榕
仲磊
纪阳
胡秋辉
赵立艳
TIAN Jiangrong;ZHONG Lei;JI Yang;HU Qiuhui;ZHAO Liyan(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;School of Food Science and Engineering,Hainan University,Haikou 570228,China;Collaborative Innovation Center for Modern Grain Circulation and Safety,Jiangsu Province Engineering Research Center of Edible Fungus Preservation and Intensive Processing,College of Food Science and Engineering,Nanjing University of Finance and Economics,Nanjing 210023,China)
出处
《食品科学》
北大核心
2025年第11期94-104,共11页
Food Science
基金
国家自然科学基金青年科学基金项目(32202037)
江苏省基础研究计划(自然科学基金)资助项目(BK20221021)
海南省自然科学基金青年基金项目(325QN237)。
关键词
杏鲍菇
活性多肽
免疫调节
分子对接
Pleurotus eryngii
active peptides
immunoregulation
molecular docking
