猪霍乱沙门菌胞外产物的核酸酶活性及其对巨噬细胞胞外诱捕网形成的影响  

The Nuclease Activity of Extracellular Products from Salmonella Choleraesuis and Its Effect on the Formation of Macrophages Extracellular Traps

作　　者：李琦 牛俊辉 王晓利[2] 毛静 全映颖 刘桂坤 雷显前 朱鹏瑶 廖成水[1] LI Qi;NIU Junhui;WANG Xiaoli;MAO Jing;QUAN Yingying;LIU Guikun;LEI Xianqian;ZHU Pengyao;LIAO Chengshui(College of Animal Science and Technology/Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Henan University of Science and Technology,Luoyang 471023,China;School of Basic Medical Sciences,Henan University of Science and Technology,Luoyang 471023,China)

机构地区：[1]河南科技大学,动物科技学院/洛阳市活载体生物材料与动物疫病防控重点实验室,洛阳471023 [2]河南科技大学基础医学院,洛阳471023

出　　处：《畜牧兽医学报》2021年第3期733-741,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31802159);河南科技大学大学生研究训练计划(2020359);河南省科技攻关计划(182102110061);河南科技大学博士启动基金项目(13480071)。

摘　　要：旨在探究猪霍乱沙门菌胞外产物的核酸酶活性及其对巨噬细胞胞外诱捕网形成的影响。采用平板培养法、琼脂糖凝胶电泳法和琼脂扩散法观察猪霍乱沙门菌胞外产物降解λDNA的活性,同时,分析不同温度、pH和金属离子对胞外产物核酸酶活性的影响以及胞外产物对巨噬细胞胞外诱捕网形成的影响。猪霍乱沙门菌胞外产物具有降解λDNA的DNA酶活性,高温和酸性条件抑制DNA酶的活性,活性最适温度和pH分别为42℃和9.0,且胞外产物的核酸酶可被60℃以上的温度灭活。添加Na^(+)、K^(+)、Ca^(2+)和Ni^(2+)对胞外产物切割λDNA的活性没有影响;5.00和10.00 mmol·L^(-1)的Ba^(2+)、Mg^(2+)和Mn^(2+)可提高胞外产物的DNA酶活性;0.01~1.00 mmol·L^(-1)的Zn^(2+)、Cu^(2+)和Fe^(3+)对胞外产物的DNA酶活性具有促进作用,但Co^(2+)则表现出抑制作用。荧光显微镜和定量分析均显示,猪霍乱沙门菌不能刺激巨噬细胞释放胞外诱捕网,而猪霍乱沙门菌胞外产物可显著降解白色念珠菌刺激巨噬细胞形成的胞外诱捕网。本研究证实了猪霍乱沙门菌胞外产物的DNA酶活性,为进一步探究胞外产物在猪霍乱沙门菌感染与宿主免疫互作中的作用提供参考。This study aimed to explore the nuclease activity of extracellular products from Salmonella Choleraesuis and its effect on the formation of macrophages extracellular traps(METs).Agar culture assay,agarose gel electrophoresis,and agar diffusion method were used to investigate the nuclease activity of extracellular products of S.Choleraesuis.Effects of temperature,pH,and metal ions on the nuclease activity of extracellular products were explored by agarose gel electrophoresis.The effect of the extracellular products on METs induced by Candida albicans was measured.The results showed that λDNA was degraded by the extracellular products from S.Choleraesuis.The nuclease activity was inhibited under a high temperature and acidic conditions.The optimum temperature and pH of the nuclease activity were 42℃and pH 9.0,respectively.The nuclease of the extracellular products was inactivated under a high temperature of 60℃or higher.Na^(+),K^(+),Ca^(2+),and Ni^(2+)did not affect the nuclease activity.The high ionic strength of Ba^(2+),Mg^(2+),and Mn^(2+)with 5.00 and 10.00 mmol·L^(-1) enhanced the nuclease activity of extracellular products.The activity of cleaveλDNA promoted with the concentrations of 0.01-1.00 mmol·L^(-1) of Zn^(2+),Cu^(2+),and Fe^(3+);conversely,low concentrations of 0.01-1.00 mmol·L^(-1) of Co^(2+)inhibited the nuclease activity.Fluorescence microscope and quantification analyses revealed that the METs induced by Candida albicans were degraded by the extracellular products from S.Choleraesuis.This study confirmed that the extracellular products from S.Choleraesuis have nuclease activity,which provides a reference for further study of the role of extracellular products in the interaction between S.Choleraesuis infection and the host immune.

关 键 词：猪霍乱沙门菌 胞外产物 核酸酶 巨噬细胞 胞外诱捕网 

分 类 号：S852.612[农业科学—基础兽医学]