[1]张 琳,陈 莹,朱可蒙,等.鸡毒支原体GroEL蛋白通过NF-κB信号通路诱导DF-1细胞释放IL-1β的研究[J].中国预防兽医学报,2019,(03):223-228.[doi:0.3969/j.issn.1008-0589.201808032]
 ZHANG Lin,CHEN Ying,ZHU Ke-meng,et al.NF-κB signaling pathway regulates IL-1β production in DF-1 cells exposed to Mycoplasma gallisepticum GroEL protein[J].Chinese journal of preventive veterinary medicine,2019,(03):223-228.[doi:0.3969/j.issn.1008-0589.201808032]





NF-κB signaling pathway regulates IL-1β production in DF-1 cells exposed to Mycoplasma gallisepticum GroEL protein

张 琳1陈 莹2朱可蒙1赵雅芝1潘 巧1郝文君1于 颖1*辛九庆1*

 (1. 中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室,黑龙江 哈尔滨 150069;
2. 东北农业大学 动物医学学院,黑龙江 哈尔滨 150030)

ZHANG Lin1 CHEN Ying2 ZHU Ke-meng1 ZHAO Ya-zhi1 PAN Qiao1 HAO Wen-jun1 YU Ying1* XIN Jiu-qing1*

 (1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150069, China; 2 College of Veterinary Medicine, Northeast Agriculture University, Harbin 150030, China)
Mycoplasma gallisepticum  GroEL  IL-1β  p65  NF-κB
鸡毒支原体(MG)脂质相关膜蛋白(LAMPs)在刺激宿主细胞天然免疫中起重要作用,GroEL蛋白是本实验室前期利用质谱鉴定筛选出的LAMPS中的关键组分。为研究GroEL蛋白诱导宿主细胞炎性反应的信号通路,本研究通过原核表达系统表达出MG的GroEL蛋白,利用激光共聚焦试验、荧光定量PCR和western blot方法分别检测GroEL蛋白的粘附特性、p65入核、磷酸化水平及IL-1β分泌情况。激光共聚焦试验结果显示,GroEL蛋白能够黏附于DF-1细胞表面并刺激DF-1细胞中p65从胞浆转入细胞核;western blot检测显示GroEL蛋白能够激活NF-κB信号通路促进p65磷酸化水平提高;荧光定量PCR检测显示GroEL蛋白能够促进IL-1β释放,当NF-κB信号通路被抑制后,IL-1β的释放显著下降(p<0.01)。本研究结果表明MG GroEL蛋白能够粘附于宿主细胞表面并通过激活NF-κB信号通路诱导宿主细胞IL-1β的释放,从而在炎症反应中发挥作用。本研究为深入研究MG致病机制提供了实验依据。
The lipid associated membrane proteins (LAMPs) of Mycoplasma gallisepticum (MG) play important roles in stimulating innate immunity of host cells, in the previous study, we have identified the GroEL protein is the key component of LAMPs by mass spectrometry analysis. In order to study the signaling pathway of host cell inflammatory reaction induced by GroEL protein, the GroEL protein was expressed in prokaryotic expression system, and the adhere property of GroEL protein, the p65 translocated into nuclear, the phosphorylation level of p65 and the release of IL-1β in DF-1 cells were detected by the confocal laser scanning microscopy, SYBR Green I real-time PCR, and western blot. The confocal laser scanning microscopy results showed that GroEL protein was able to adhere to the surface of DF-1 cells and stimulated the p65 translocated into nuclear. Western blot results showed the stimulation of GroEL protein activated the NF-κB signaling pathway and promote the phosphorylation level of p65. SYBR Green I real-time PCR detection showed GroEL protein promoted the release of IL-1β inDF-1 cells. While the NF-κB signaling pathway was inhibited, and the mRNA transcriptional level of IL-1β decreased significantly. These results suggested that GroEL protein of MG had the ability to adhere to the surface of DF-1 cells and play a role in inflammatory reaction by the release of IL-1β activated via the activation of NF-κB signaling pathway, and provided information for further study the pathogenic mechanism of MG.


[1]Reddy S K, Singh P, Silim A, et al. Lymphoproliferative responses of specific-pathogen-free chickens to Mycoplasma gallisepticum strain PG31 [J]. Avian Pathol, 1998, 27(3): 277-283.
[2]Stipkovits L, Kempf I. Mycoplasmoses in poultry [J]. Rev Sci Tech, 1996, 15(4): 1495-1525.
[3]Yu Ying, Chen Ying, Wang Yang, et al. TLR2/MyD88/NF-κB signaling pathway regulates IL-1β production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs [J]. Microb Pathog, 2018, 117: 225-231.
[4]Ricci C, Ortore M G, Vilasi S, et al. Stability and disassembly properties of human na?觙ve Hsp60 and bacterial GroEL chaperonins. [J]. Biophys Chem, 2016, 208: 68-75.
[5]Calderwood S K, Mambula S S, Gray J R P J. Extracellular heat shock proteins in cell signaling and immunity [J]. Ann N Y Acad Sci, 2007, 1113: 28-39.
[6]Breloer M, Dorner B, Moré S H, et al. Heat shock proteins as "danger signals": Eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells [J]. Eur J Immunol, 2001, 31(7): 2051-2059.
[7]Schmitt E, Gehrmann M, Brunet M, et al. Intracellular and extracellular functions of heat shock proteins: Repercussions in cancer therapy [J]. J Leukoc Biol, 2007, 81(1): 15-27.
[8]Davis K L, Wise K S. Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans [J]. Infect Immun, 2002, 70(3): 1129-1135.
[9] Jenkins C, Geary S J, Gladd M, et al. The Mycoplasma gallisepticum OsmC-like protein MG1142 resides on the cell surface and binds heparin[J]. Microbiology, 2007, 153(5): 1455-1463.
[10]Noss E H, Pai R K, Sellati T J, et al. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis [J]. Immunology, 2001, 167(2): 910-918.
[11]Giotis E S, Ross C S, Robey R, et al. Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells [J]. Sci Rep, 2017, 7(1): 17485-17499.
[12]Indikova I, Much P, Spitkovits L, et al. Role of the GapA and CrmA cytadhesins of Mycoplasma gallisepticum in promoting virulence and host colonization [J]. Infect Immun, 2013, 81(5): 1618-1624.
[13]周长平,陈莹,李媛,等. 鸡毒支原体热休克蛋白GrpE粘附特性的鉴定[J]. 中国预防兽医学报,2017,1(1):28-33.
[14]Eskra L, Mathison A, Splitter G. Microarray analysis of mRNA levels from RAW264.7 macrophages infected with Brucella abortus [J]. Infect Immun, 2003, 71(25): 1125-1133.
[15]Arthur S, Ley S. Mitogen-activated protein kinase in innate immunity [J]. Nature, 2013, 13(26): 679-692.
[16]Gilmore T D. Introduction to NF-kappaB: Players, pathways, perspectives [J]. Oncogene, 2006, 25(51): 6680-6684.
[17]Ghosh S, May M J, Kopp E B. NF-κB and Rel proteins: Evolutionarily conserved mediators of immune responses [J]. Annu Rev Immunol, 1998, 16: 225-260.
[18]He Jun, You Xiao-xing, Zeng Yan-hua, et al. Mycoplasma genitalium-derived lipid-associated membrane proteins activate NF-kappaB through toll-like receptors 1, 2, and 6 and CD14 in a MyD88-dependent pathway [J]. Clin Vaccine Immunol, 2009, 16(12): 1750-1757.
[19]Wang Yang, Liu Su-li, Li Yuan, et al. Mycoplasma bovis-derived lipid-associated membrane proteins activate IL-1β production through the NF-κB pathway via toll-like receptor 2 and MyD88 [J]. Dev Comp Immunol, 2016, 55: 111-118.


 ZHOU Yun-lei#,LI Jian#,WEI Fei-long,et al.Development of an indirect ELISA for detecting antibodies against Mycoplasma gallisepticum based on recombinant PvpA protein[J].Chinese journal of preventive veterinary medicine,2011,(03):713.
[2]胡美容,陈鸿军,于圣青,等.鸡毒支原体穿梭质粒的构建[J].中国预防兽医学报,2012,(08):619.[doi:doi: 10.3969/j.issn.1008-0589.2012.08.07]
 HU Mei-rong,CHEN Hong-jun,YU Sheng-qing,et al.Construction of shuttle vector for Mycoplasma gallisepticum[J].Chinese journal of preventive veterinary medicine,2012,(03):619.[doi:doi: 10.3969/j.issn.1008-0589.2012.08.07]
[3]冯秋霖,刘茂军,祁 芳,等.表达鸡毒支原体TM1蛋白基因重组新城疫病毒的构建及其免疫原性研究[J].中国预防兽医学报,2013,(10):779.[doi:10.3969/j.issn.1008-0589.2013.10.01]
 FENG Qiu-lin,LIU Mao-jun,QI Fang,et al.Construction of recombinant Newcastle disease virus expressing TM1 gene of Mycoplasma gallisepticum and its immune response in chickens[J].Chinese journal of preventive veterinary medicine,2013,(03):779.[doi:10.3969/j.issn.1008-0589.2013.10.01]
 HU Si-shun,WANG Xiao-li,LU Shi-juan,et al.The immunity of Mycoplasma gallisepticum strain HS recombinant adhesion protein rpMGA1.2[J].Chinese journal of preventive veterinary medicine,2015,(03):225.[doi:10.3969/j.issn.1008-0589.2015.03.15]
[5]孙俊颖,刘志成,孙敏华,等.鸡毒支原体SYBR Green I荧光定量PCR检测方法的建立[J].中国预防兽医学报,2015,(12):938.[doi:10.3969/j.issn.1008-0589.2015.12.10]
 SUN Jun-ying,LIU Zhi-cheng,SUN Min-hua,et al.Development of a SYBR Green I real-time PCR assayfor detection of Mycoplasma gallisepticum[J].Chinese journal of preventive veterinary medicine,2015,(03):938.[doi:10.3969/j.issn.1008-0589.2015.12.10]
[6]高利萍,李 媛,周长平,等.鸡毒支原体S6株P25蛋白粘附特性的研究[J].中国预防兽医学报,2016,(02):105.[doi:10.3969/j.issn.1008-0589.2016.02.05]
 GAO Li-ping,LI Yuan,ZHOU Chang-ping,et al.The adhesive character of P25 protein in Mycoplasma gallisepticum S6 strain[J].Chinese journal of preventive veterinary medicine,2016,(03):105.[doi:10.3969/j.issn.1008-0589.2016.02.05]
 WANG Li-li,YU Yan-chao,ZHOU Chang-ping,et al.The evaluation and application of the heat shock protein DnaK of Mycoplasma gallisepticum as coating antigen in indirect ELISA[J].Chinese journal of preventive veterinary medicine,2018,(03):795.[doi:0.3969/j.issn.1008-0589.201711042]

更新日期/Last Update: 2019-04-25