小鼠IL-1β原核表达及其与在布鲁氏杆菌侵染巨噬细胞中差异表达的初步研究

doi:10.14088/j.cnki.issn0439-8114.2022.06.021

湖北农业科学 ›› 2022, Vol. 61 ›› Issue (6): 112-116.doi: 10.14088/j.cnki.issn0439-8114.2022.06.021

小鼠IL-1β原核表达及其与在布鲁氏杆菌侵染巨噬细胞中差异表达的初步研究

杨馨, 邹德颖, 张凯, 常江, 常恒祯, 战俊澎, 郭珣, 刘益辛, 程梦妍, 韩成, 胡盼, 卢士英, 李岩松, 柳增善, 任洪林

吉林大学人兽共患病研究教育部重点实验室/人兽共患病研究所/动物医学学院,长春 130062

收稿日期:2021-12-09 出版日期:2022-03-25 发布日期:2022-04-18
通讯作者: 任洪林（1974-）,男,教授,博士,博士生导师,主要从事人兽共患细菌病、食品安全快检技术及分子生物学与毒理学研究,（电子信箱）renhl@jlu.edu.cn。
作者简介:杨馨（1995-）,女,陕西西安人,在读硕士研究生,研究方向为兽医公共卫生学,（电话）18392364878（电子信箱）1971908850@qq.com。
基金资助:
吉林省科技发展计划资助项目（20200402059NC）; 中央高校基本科研业务费专项资金资助项目（45121031B007）

Prokaryotic expression of mouse IL-1β and preliminary study on its differential expression in Brucella infection

YANG Xin, ZOU De-ying, ZHANG Kai, CHANG Jiang, CHANG Heng-zhen, ZHAN Jun-peng, GUO Xun, LIU Yi-xin, CHENG Meng-yan, HAN Cheng, HU Pan, LU Shi-ying, LI Yan-song, LIU Zeng-shan, REN Hong-lin

Key Laboratory of Zoonosis Research, Ministry of Education/Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, China

Received:2021-12-09 Online:2022-03-25 Published:2022-04-18

摘要/Abstract

摘要： 为研究IL-1β在布鲁氏杆菌（Brucella）侵染小鼠巨噬细胞过程中的表达情况,首先构建重组小鼠IL-1β原核稳定表达系统,免疫新西兰兔获得多克隆抗体,通过ELISA检测血清效价,并通过Western blot分析其活性,再以感染复数（multiplicity of infection,MOI）为1∶100（细菌∶细胞）建立猪种布鲁氏杆菌S2株侵染小鼠巨噬细胞RAW264.7的模型,利用RT-PCR检测细菌侵染过程中IL-1β和MyD88 mRNA的变化水平;Western blot分析IL-1β在蛋白水平的变化。结果表明,重组蛋白IL-1β分子质量31 ku,多克隆抗体效价为1∶256 000,并且具有良好的特异性;与对照组相比,感染后IL-1β mRNA和IL-1β蛋白表达上调,胞内细菌数减少。猪种布鲁氏杆菌S2株侵染巨噬细胞IL-1β的表达量随时间变化并影响胞内活菌数。

关键词: 布鲁氏杆菌（Brucella）, 原核表达, 多克隆抗体, IL-1β, MyD88

Abstract: To explore the expression of IL-1β during the infection of host cells by Brucella, the prokaryotic expression of IL-1β in mouse was constructed, polyclonal antibodies was obtained by immunization, and its activity was detected by ELISA and Western blot. The 1∶100 （bacterial∶cell） multiplicity of infection was then used to model the infection of RAW264.7 macrophages by Brucella suis S2. Expression of IL-1β mRNA and MyD88 mRNA was detected by RT-PCR; IL-1β protein was analyzed by Western blot. The molecular weight of the recombinant protein was about 31 ku. And the titer of the polyclonal antibody prepared by ELISA was 1∶25 600, with good immunoreactivity and specificity. The expression of IL-1β mRNA and IL-1β protein increased after infection. And the number of intracellular bacteria decreased. The expression of IL-1β in macrophages infected with Brucella suis S2 varied over time and affected the intracellular bacteria。

Key words: Brucella, prokaryotic expression, polyclonal antibody, IL-1β, MyD88

中图分类号:

R392

杨馨, 邹德颖, 张凯, 常江, 常恒祯, 战俊澎, 郭珣, 刘益辛, 程梦妍, 韩成, 胡盼, 卢士英, 李岩松, 柳增善, 任洪林. 小鼠IL-1β原核表达及其与在布鲁氏杆菌侵染巨噬细胞中差异表达的初步研究[J]. 湖北农业科学, 2022, 61(6): 112-116.

YANG Xin, ZOU De-ying, ZHANG Kai, CHANG Jiang, CHANG Heng-zhen, ZHAN Jun-peng, GUO Xun, LIU Yi-xin, CHENG Meng-yan, HAN Cheng, HU Pan, LU Shi-ying, LI Yan-song, LIU Zeng-shan, REN Hong-lin. Prokaryotic expression of mouse IL-1β and preliminary study on its differential expression in Brucella infection[J]. HUBEI AGRICULTURAL SCIENCES, 2022, 61(6): 112-116.

参考文献

[1] GARLANDA C, DINARELLO C A, MANTOVANI A.The interleukin-1 family: Back to the future[J]. Immunity,2013,39(6):1003-1018.
[2] PRISCILA C C, MARCOT R G, FÁBIO A V M, et al. Brucellaabortus nitric oxide metabolite regulates inflammasome activation and IL-1β secretion in murine macrophages[J]. European journal of immunology, 2019,49(7): 1023-1037.
[3] MEDZHITOV R,PRESTON-HURLBURT P,KOPP E,et al.MyD88 is an adaptor protein in the Toll/IL-1 receptor family signaling pathways[J]. Molecular cell, 1998, 2(2):253-258.
[4] CUTLER S J, WHATMORE A M, COMMANDER N J.Brucellosis-new aspects of an old disease[J]. Journal of applied microbiology, 2010, 98(6):1270-1281.
[5] SKENDROS P, BOURA P.Immunity to brucellosis[J]. Revue Scientifique Et Technique, 2013, 32(1):137-47.
[6] 郭兴. 绵羊白介素1β及其受体拮抗因子全长cDNA克隆分子特性及差异表达分析[D].长春:吉林大学, 2015.
[7] CELLI J.The intracellular life cycle of Brucella spp.[J].Microbiology spectrum, 2019, 7(2):7-23.
[8] 耿梦杰,任翔,彭质斌,等. 2021年我国夏季重点传染病疫情形势分析[J].疾病监测,2021,36(8):811-817.
[9] 任洪林,卢士英,周玉,等. 布鲁氏菌病的研究与防控进展[J].中国畜牧兽医, 2009, 36(9):139-143.
[10] 王功民,池丽娟,马世春,等.我国畜间布鲁氏菌病流行特点及原因分析[J].中国动物检疫,2010,27(7):62-63.
[11] BEN-SASSON S Z, HU-LI J, QUIEL J, et al. IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation[J]. Proceedings of the national academy of sciences, 2009, 106(17):7119-7124.
[12] HOP H T, ARAYAN L T, REYES A, et al.Simultaneous RNA-seq based transcriptional profiling of intracellular Brucella abortus and B.abortus-infected murine macrophages[J]. Microbial pathogenesis, 2017,113:57-67.
[13] 荆明龙, 李敏, 王小凤,等. 布鲁菌感染细胞NF-κB的表达水平检测及其干扰方法研究[J]. 石河子大学学报(自然科学版), 2018, 36(3):315-320.
[14] ALI A F, ABDELWAHAB M G.Interleukin-1β, tumor necrosis factor-α, and oxidative stress biomarkers in cows with acute Brucellaabortus infection[J].Comparative clinical pathology,2021(30):311-315.
[15] WESCHE H, HENZEL W J, SHILLINGLAW W, et al.MyD88: An adapter that recruits IRAK to the IL-1 receptor complex[J]. Immunity, 1997, 7(6):837-847.
[16] ADACHI O.Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function[J]. Immunity,1998,9(1):143-150.
[17] VICKERS T A, ZHANG H, GRAHAM M J, et al.Modification of MyD88 mRNA splicing and inhibition of IL-1β signaling in cell culture and in mice with a 2\"-O-methoxyethyl-modified oligonucleotide[J]. Journal of immunology, 2006, 176(6):3652-3661.
[18] WEISS D S, TAKEDA K, AKIRA S, et al.MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus[J]. Infection and immunity, 2005, 73(8):5137-5143.
[19] VIEIRA A, SILVA T, MOL J, et al.MyD88 and TLR9 are required for early control of Brucella ovis infection in mice[J]. Research in veterinary science, 2013, 94(3):399-405.
[20] LACEY C A,MITCHELL W J, BROWN C R, et al.Temporal role for MyD88 in a model of Brucella-induced arthritis and musculoskeletal inflammation[J]. Infection and immunity,2017,85(3): e00961-16.

小鼠IL-1β原核表达及其与在布鲁氏杆菌侵染巨噬细胞中差异表达的初步研究

Prokaryotic expression of mouse IL-1β and preliminary study on its differential expression in Brucella infection

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