马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响

doi:10.11843/j.issn.0366-6964.2020.10.026

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (10): 2576-2583.doi: 10.11843/j.issn.0366-6964.2020.10.026

马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响

陈楷文, 华承薇, 袁宸, 潘飞, 蔺辉星, 范红结, 马喆*

南京农业大学动物医学院, 南京 210095

收稿日期:2020-03-02 出版日期:2020-10-25 发布日期:2020-10-26
通讯作者: 马喆,主要从事猪链球菌病病原马链球菌兽疫亚种(SEZ)和畜禽源沙门菌的致病机理以及防控技术研究,E-mail:mazhe@njau.edu.cn
作者简介:陈楷文(1998-),男,江苏扬州人,本科生,主要从事畜禽病原微生物研究,E-mail:17116420@njau.edu.cn
基金资助:
国家自然科学基金（31973004；31772746）

Effects of Enolase of Streptococcus equi ssp. zooepidemicus on Phagocytic Functions of Alveolar Macrophages in Mice

CHEN Kaiwen, HUA Chengwei, YUAN Chen, PAN Fei, LIN Huixing, FAN Hongjie, MA Zhe*

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2020-03-02 Online:2020-10-25 Published:2020-10-26

摘要/Abstract

摘要： 旨在研究马链球菌兽疫亚种（Streptococcus equi ssp.zooepidemicus，SEZ）烯醇化酶（enolase，Eno）对小鼠肺泡巨噬细胞（RAW264.7）吞噬能力的影响。通过构建原核表达质粒获得重组烯醇化酶（rEno），采用台盼蓝活细胞计数法，判定在不同处理浓度和时间下rEno蛋白对RAW264.7细胞的细胞毒性。将rEno蛋白与RAW264.7细胞共孵育后，用SEZ作用于细胞并检测细胞吞菌数量，判断RAW264.7细胞对SEZ的吞噬活性。进一步通过活细胞稳定同位素标记技术（SILAC）和蛋白质谱分析技术（LC-MS/MS），筛选到RAW264.7细胞中可能与SEZ Eno存在相互作用的候选蛋白。结果发现，10 μg·mL^-1 rEno蛋白处理对RAW264.7细胞有明显的细胞毒性，且10 μg·mL^-1 rEno蛋白处理RAW264.7细胞2和4 h可显著抑制其对SEZ的吞噬作用（P<0.01、P<0.05）。初步筛选到RAW264.7细胞中动力蛋白激活蛋白亚单位蛋白（dynactin subunit protein 2，Dctn）、整合素α-M蛋白（integrin alpha-M）等17种可能与Eno发生互作的蛋白。本研究获得了rEno重组表达蛋白，发现rEno可减少RAW264.7细胞对SEZ的吞噬，互作蛋白的初步筛选也为进一步揭示Eno在SEZ抗吞噬中的作用机制奠定了基础。

关键词: 马链球菌兽疫亚种, 烯醇化酶, RAW264.7细胞, 吞噬作用, 蛋白互作

Abstract: To investigate the effect of enolase (Eno) of Streptococcus equi ssp. zooepidemicus (SEZ) on phagocytosis of mouse alveolar macrophages (RAW264.7). Recombinant enolase (rEno) was obtained by constructing prokaryotic expression plasmid, and the cytotoxicity of rEno protein on RAW264.7 cell proliferation was determined by trypan-blue living cell count method. After the rEno protein was incubated with RAW264.7 cells, SEZ was applied to the cells and the quantity of bacteria being phagocytosed was detected to determine the phagocytic activity of RAW264.7 cells. Further, candidate proteins that might interact with SEZ Eno in RAW264.7 cells were screened by live cell stable isotope labeling (SILAC) and protein spectrum analysis (LC-MS/MS). It was found that protein treatment (rEno，10 μg·mL^-1) had significant cytotoxic effects on RAW264.7 cells. Treatment of RAW264.7 cells with 10.0 μg·mL^-1 rEno protein for 2 and 4 hours could significantly inhibit the phagocytosis of RAW264.7 cells (P<0.01, P<0.05). In RAW264.7 cells, dynactin subunit protein 2 (Dctn), integrin alpha-M and about 17 proteins that might interact with Eno were preliminarily identified as rEno interaction proteins. The rEno recombinant expression protein was obtained in this study, and it could reduce the phagocytosis of RAW264.7 cells to SEZ. Preliminary screening of interacting proteins also laid a foundation for further revealing the mechanism of Eno in the anti-phagocytosis of SEZ.

Key words: SEZ, enolase, RAW264.7 cell, phagocytosis, interactions between protein

中图分类号:

S852.61

陈楷文, 华承薇, 袁宸, 潘飞, 蔺辉星, 范红结, 马喆. 马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响[J]. 畜牧兽医学报, 2020, 51(10): 2576-2583.

CHEN Kaiwen, HUA Chengwei, YUAN Chen, PAN Fei, LIN Huixing, FAN Hongjie, MA Zhe. Effects of Enolase of Streptococcus equi ssp. zooepidemicus on Phagocytic Functions of Alveolar Macrophages in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2576-2583.

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马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响

Effects of Enolase of Streptococcus equi ssp. zooepidemicus on Phagocytic Functions of Alveolar Macrophages in Mice

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