猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应

doi:10.11843/j.issn.0366-6964.2014.11.018

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (11): 1866-1873.doi: 10.11843/j.issn.0366-6964.2014.11.018

猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应

刘建涛，张强，宋娅静，闫树仙，于君平，张安定，金梅林^*

（华中农业大学农业微生物国家重点实验室，武汉 430070）

收稿日期:2014-04-08 出版日期:2014-11-23 发布日期:2014-11-24
通讯作者: 金梅林，E-mail：jml8328@126.com
作者简介:刘建涛(1988-)，男，河南驻马店人，硕士，主要从事猪链球菌2型致病研究，E-mail：liujiantao.ok@163.com
基金资助:
国家“十二五”高技术研究发展计划（863）项目（2011AA10A210）；国家重点基础研究发展计划计划(973)项目(2012CB518805)

Streptococcus suis Serotype 2 Protein AroC Induces Toll-Like Receptor 4-Dependent Inflammatory Responses in RAW264.7 via p38MAPK and NF-κB Signaling

LIU Jian-tao，ZHANG Qiang，SONG Ya-jing，YAN Shu-xian，YU Jun-ping，ZHANG An-ding，JIN Mei-lin^*

(National Key Laboratory of Agricultural Microbiology，Huazhong Agricultural University,Wuhan 430070，China)

Received:2014-04-08 Online:2014-11-23 Published:2014-11-24

摘要/Abstract

摘要：

在大肠杆菌中表达与纯化猪链球菌表面蛋白分支酸合成酶，研究其对小鼠巨噬细胞RAW264.7细胞分泌细胞因子的影响，并试图解释其分子机制。以SC19基因组为模板，PCR扩增aroC基因，构建表达质粒，转化到BL21（DE3）中并诱导表达，所得融合蛋白主要以包涵体的形式存在，以8 mol• L^-1尿素（含有50 mmol•L^-1 Tris，pH8.0）为变性剂，对构建于pET-28a（+）的猪链球菌aroC基因在大肠杆菌BL21中表达的包涵体进行变性、复性、纯化、去除LPS以及除菌。以aroC蛋白体外刺激RAW264.7细胞，共同孵育4 h后用real-time PCR的方法分析IL-1β、TNF-α的mRNA转录量。用ERK1/2、JNK、NF-κB和P38的抑制剂以及TLR2和TLR4的特异性抗体来解释其引起炎性反应的分子基础。结果显示成功对aroC蛋白进行了变性、复性及纯化，该蛋白具有刺激巨噬细胞表达细胞因子的能力，用NF-κB的抑制剂以及TLR4的特异性抗体预处理细胞后能明显降低aroC导致的IL-1β、TNF-α mRNA的转录，用P38的抑制剂预处理细胞后能明显降低aroC导致的TNF-α mRNA的转录量。结果证明aroC在RAW264.7中通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应。

关键词: 猪链球菌, 分支酸合成酶, 细胞因子, p38MAPK, NF-&kappa, B, TLR4

Abstract:

Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis.The purpose of this study was to express and purify protein aroC of S.suis and to analyse its ability of stimulating RAW264.7 cells inflammatory factor expression and to explain its molecular mechanism.aroC gene was amplified by using the genome of SC19 and cloned into the expression plasmid pET28a(+).The inclution bodies of recombinant aroC were expressed in E.coli of BL21.It was dissolved with 8 mol•L^-1 urea （50 mmol•L^-1 Tris，pH 8.0），and then renaturalized by dialysis and purified.The recombinant LPS-free and germ free protein was further tested on the stimulation of cytokine of RAW264.7 by real-time PCR.In order to analyse its molecular mechanism of stimulating macrophage inflammatory factors，we pre-treated the cell cultures with specific ERK1/2，JNK，NF-κB and P38 pathway inhibitors and antibodies of TLR2 and TLR4.Results showed that the aroC protein was renaturalized and purified successfully.It has a strong stimulating on secretion of cytokines of RAW264.7，and pre-treatment of the cell cultures with specific NF-κB pathway inhibitors and TLR4 antibody significantly decreased aroC induced TNF-α and IL-1β mRNA production，and pre-treatment of the cell cultures with specific p38 pathway inhibitors significantly decreased aroC induced TNF-α mRNA production.The results presented here demonstrate that protein aroC of S.suis serotype 2 induces Toll-like receptor 4-dependent inflammatory responses in RAW264.7 via p38MAPK and NF-κB Signaling.

Key words: Streptococcus suis, aroC, cytokine, p38MAPK, NF-κB, TLR4

中图分类号:

S852.61

刘建涛，张强，宋娅静，闫树仙，于君平，张安定，金梅林. 猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应[J]. 畜牧兽医学报, 2014, 45(11): 1866-1873.

LIU Jian-tao，ZHANG Qiang，SONG Ya-jing，YAN Shu-xian，YU Jun-ping，ZHANG An-ding，JIN Mei-lin. Streptococcus suis Serotype 2 Protein AroC Induces Toll-Like Receptor 4-Dependent Inflammatory Responses in RAW264.7 via p38MAPK and NF-κB Signaling[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1866-1873.

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猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应

Streptococcus suis Serotype 2 Protein AroC Induces Toll-Like Receptor 4-Dependent Inflammatory Responses in RAW264.7 via p38MAPK and NF-κB Signaling

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