堆型艾美耳球虫外分泌蛋白致细胞损伤研究  

Study of cell damage under exocrine protein of Eimeria acervulina

作　　者：王黎霞[1] 黄芳[2] 张建军[2] 安健[2] WANG Lixia;HUANG Fang;ZHANG Jianjun;AN Jian(College of Animal Science and Veterinary,Beijing Vocational College of Agriculture,Beijing 102442,China;College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农业职业学院牧医系,北京102442 [2]北京农学院动物科学技术学院,北京102206

出　　处：《北京农学院学报》2021年第1期100-104,共5页Journal of Beijing University of Agriculture

基　　金：北京农业职业学院院级科研项目(XY-HT-19-03);北京市特色高水平骨干专业群项目-动物医学专业群项目-技术平台与社会服务建设项目(PXM2020-157102-000060)。

摘　　要：【目的】研究堆型艾美耳球虫(Eimeria acervulina)外分泌蛋白是否具有外毒素和致细胞损伤作用。【方法】将牛肾传代细胞与E.acervulina子孢子共培养2.5 h,更换无血清培养液,每间隔12 h收集入侵后0~60 h的该时间点前12 h所有培养液并透析浓缩,SDS-PAGE和Western Blot检测,确定E.acervulina的外分泌蛋白,将此蛋白加培养液培养牛肾传代细胞,利用吖啶橙和碘化丙啶染色,判断细胞损伤。【结果】E.acervulina分泌约70 kD的蛋白,12~24 h分泌蛋白最多,24~36 h蛋白分泌量明显下降,36~48 h以后几乎看不到蛋白带,0~12 h和48~60 h看不到外分泌蛋白,用含该蛋白的培养液培养牛肾传代细胞,细胞膜和核膜的通透性增加,【结论】E.acervulina第一代裂殖生殖时分泌约70 kD的蛋白,该蛋白损伤细胞膜和核膜,证明该蛋白具有球虫毒素的功能。【Objective】The aim of the study is to investigate whether the exocrine protein of E.acervulina injure the host cell.【Methods】Madin-Darby bovine kidney cells were co-cultured with sporozoite of E.acervulina for 2.5 hours in six-well plate,then the culture media was replaced by no serum medium.The medium was collected every 12 hours from 12-48 hours.The coccidian exocrine protein was identificated by SDS-PAGE and Western Blot.The pathogenicity of the protein was identificated by acidine orange and propidium iodide.【Results】The results indicated a 70 kD protein was primarily secrated by E.acervulina from 12 hours to 24 hours after invasion,which could increase the permeability of the cell and nuclear membrane and injure Madin-Darby bovine kidney cells.【Conclusion】A 70 kD protein was found to be exocrine protein of E.acervulina,whose function was exogenous toxin to host cell。

关 键 词：堆型艾美耳球虫 体外培养 外分泌蛋白 细胞损伤 

分 类 号：S851.34[农业科学—预防兽医学]