基于LpxL和LpxM的APEC感染鸡中MIF的作用及其机理研究
结题报告
批准号:
31972711
项目类别:
面上项目
资助金额:
57.0 万元
负责人:
高崧
依托单位:
学科分类:
兽医传染病学
结题年份:
2023
批准年份:
2019
项目状态:
已结题
项目参与者:
高崧
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中文摘要
禽致病性大肠杆菌(APEC)类脂A酰基转移酶LpxL、LpxM突变株E058 ΔlpxL ΔlpxM感染SPF鸡后12小时起,血清中巨噬细胞游走抑制因子(MIF)的蛋白浓度持续高于亲本株感染鸡,推测MIF可能参与鸡抵抗APEC感染的应答。通过沉默、拯救HD11细胞MIF蛋白,阐明MIF在鸡巨噬细胞吞噬、杀伤APEC中的作用;通过亲本株、突变株分别感染HD11、鸡外周血单核细胞(PBMC),监测细胞培养上清中MIF浓度、细胞质中ERK1/ERK2通路的激活及促炎症、抗炎症细胞因子的产生,明确APEC亲本株、突变株感染后MIF的来源细胞、败血症发生的可能机制;在亲本株、突变株感染SPF鸡时,给予重组鸡MIF或鸡MIF特异性抗体,找出MIF在体内参与抵抗APEC感染的直接证据。本研究有利于厘清MIF在APEC感染中的作用,为禽大肠杆菌病的防制提供新思路。
英文摘要
In 1-day-old specific pathogen free (SPF) chicken, 50% lethal dose (LD50) of avian pathogenic Escherichia coli (APEC) strain E058 was 104.75, while the LD50 of the mutant E058ΔlpxLΔlpxM was 1010.30. Sepsis was observed in 14-day-old SPF birds affected with APEC E058 strain 24 hous post-challenge and deaths began thereafter. However, SPF chickens challenged with the mutant E058ΔlpxLΔlpxM kept healthy till ten days post-challenge. Bacterial phagocytosis and bacterial killing of mutant E058ΔlpxLΔlpxM by chicken macrophage cell line HD11 was higher than those of parent strain E058. The level of macrophage migration inhibitory factor (MIF) in serum of 14-day-old SPF chickens challenged with the mutant E058ΔlpxLΔlpxM was persistently higher than that of birds challenged with the parent strain since 12 hours post-challenge, and the difference was extremely significant at 24 hours post-challenge (p<0.0001). It was implied that MIF played an important role in chicken response to APEC infection. MIF silencing macrophage HD11 using MIF small interfering RNA (siRNA), and MIF silencing HD11 compensated with the recombinant chicken MIF (rchMIF) will be applied to incubate with APEC E058 and mutant E058ΔlpxLΔlpxM, respectively, then the bacterial phagocytosis and bacterial killing are analyzed to explore the effect of MIF on the bacterial phagocytosis and bacterial killing of the macrophage HD11. Macrophage HD11 and chicken peripheral blood mononuclear cells (PBMCs) are going to be infected with APEC E058, E058ΔlpxLΔlpxM mutant, respectively, MIF concentrations in cell culture supernatants are measured by ELISA to trace the source of MIF in birds against APEC infection; phosphorylated and total ERK1/2 proteins in cytosolic extracts are detected by Western blot to probe the activation of ERK1/ERK2 pathway; a panel of proinflammatory (TNF-α、IL-1βand IL-6) and antiinflammatory (IL-10、IL-20 and IL-27) cytokines by both HD11 macrophage and PBMCs stimulated with either APEC E058 strain or mutant E058ΔlpxLΔlpxM in the cell culture supernatants are measured by ELISA to clarify the potential mechanism of the sepsis caused by only APEC E058 strain while not the E058ΔlpxLΔlpxM mutant. At one hour ahead, simultaneously, and one hour behind the 14-day-old SPF chickens challenged via the air sac with either APEC E058 strain or the mutant E058ΔlpxLΔlpxM, chicken MIF specific monoclonal antibody, or soluble rchMIF protein will be injected into the birds cervical subcutaneously, a panel of proinflammatory and antiinflammatory cytokines in the serum of challenged birds is going to be monitored by ELISA; bacterial loads in challenged chicken blood and tissues will be determined by plating; the mortality of challenged birds is recorded till 10 days post-challenge. All these data will provide the evidence that MIF plays an import role to protect chickens from APEC infection. This study is of a theoretical significance that MIF is involved in chicken response to APEC infection, more importantly, MIF could be a potential attractive target for immune-modulating prevention and control of avian colibacillosis.
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DOI:--
发表时间:2022
期刊:畜牧兽医学报
影响因子:--
作者:李梦妮;王航;傅思瑶;杨梓纯;许炎辉;樊毛迪;高崧;刘秀梵
通讯作者:刘秀梵
DOI:--
发表时间:2023
期刊:中国畜牧兽医
影响因子:--
作者:程前;高清清;王雨禾;郇长超;高崧
通讯作者:高崧
DOI:--
发表时间:2022
期刊:微生物学报
影响因子:--
作者:张鹏池;陈小雅;王航;李林;蒋路遥;吴佳炎;黄恩赐;高崧;刘秀梵
通讯作者:刘秀梵
DOI:10.1016/j.micpath.2020.103990
发表时间:2020-01
期刊:Microbial pathogenesis
影响因子:3.8
作者:X. Mu;Ruxia Gao;Wenhua Xiao;Q. Gao;Chunguang Cao;Huiqinq Xu;Song Gao;Xiufan Liu
通讯作者:X. Mu;Ruxia Gao;Wenhua Xiao;Q. Gao;Chunguang Cao;Huiqinq Xu;Song Gao;Xiufan Liu
DOI:10.11843/j.issn.0366-6964.2020.12.021
发表时间:2020
期刊:畜牧兽医学报
影响因子:--
作者:薛菲;杨梓纯;许炎辉;王娅玲;喻婷;樊毛迪;刘娟华;高崧;刘秀梵
通讯作者:刘秀梵
靶向APEC OmpT裂解作用的CATH-3分子改造与杀菌机制研究
  • 批准号:
    32373011
  • 项目类别:
    面上项目
  • 资助金额:
    50万元
  • 批准年份:
    2023
  • 负责人:
    高崧
  • 依托单位:
禽致病性大肠杆菌ompT质粒编码基因的特殊致病作用及其成因研究
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    31672553
  • 项目类别:
    面上项目
  • 资助金额:
    62.0万元
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    2016
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    高崧
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    31272559
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    面上项目
  • 资助金额:
    80.0万元
  • 批准年份:
    2012
  • 负责人:
    高崧
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iucABCDiutA中单基因及其组合对APEC致病性的影响
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    30972196
  • 项目类别:
    面上项目
  • 资助金额:
    30.0万元
  • 批准年份:
    2009
  • 负责人:
    高崧
  • 依托单位:
基于APEC的UPEC毒力基因贮库的研究
  • 批准号:
    30771604
  • 项目类别:
    面上项目
  • 资助金额:
    28.0万元
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    2007
  • 负责人:
    高崧
  • 依托单位:
禽病原性大肠杆菌新致病基因的研究
  • 批准号:
    30471281
  • 项目类别:
    面上项目
  • 资助金额:
    20.0万元
  • 批准年份:
    2004
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    高崧
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  • 批准号:
    39770562
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    面上项目
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    10.0万元
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    1997
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    高崧
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禽病原性大肠杆菌外膜蛋白型与免疫保护关系的研究
  • 批准号:
    39500110
  • 项目类别:
    青年科学基金项目
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    8.0万元
  • 批准年份:
    1995
  • 负责人:
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  • 依托单位:
国内基金
海外基金