犬流感病毒诱导的Beagle犬NLRP3炎性小体相关基因表达谱  

Expression profile of NLRP3 inflammasome related genes in Beagle dogs challenged by canine influenza virus

作　　者：陶攀[1,2] 宁章勇 王贵平[2] 贾爱卿 周沛 李守军[1] 张桂红[1] TAO Pan;NING Zhangyong;WANG Guiping;JIA Aiqing;ZHOU Pei;LI Shoujun;ZHANG Guihong(College of Veterinary Medicine,South China Agricultural University,Guangzhou,510642 China;Guangdong Haida Animal Husbandry and Veterinary Research Institute,Guangzhou,511400 China)

机构地区：[1]华南农业大学兽医学院,广东广州5106422 [2]广东海大集团畜牧兽医研究院,广东广州511400

出　　处：《中国兽医学报》2022年第8期1564-1576,共13页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31672563)。

摘　　要：炎症反应是机体对抗异物的一种先天性免疫反应,NLRP3(nod-like receptor family,pyrin domain-containing 3)炎症小体途径是炎症反应的主要途径之一,在机体对抗病原微生物反应中起着重要的作用。为了深入研究犬的NLRP3炎症小体在犬感染H3N2犬流感病毒(CIV)后的变化,对未感染以及感染H3N2 CIV早期(3 d)和后期(7 d)的比格犬心脏、肝脏、脾脏、肺脏、肾脏、脑、气管和淋巴结组织进行病理学检测。结果发现,感染H3N2 CIV的比格犬,在早期没有明显的病理变化,炎症反应不明显,但后期炎症反应明显增强。通过荧光定量PCR检测比格犬感染H3N2 CIV后NLRP3、ASC、Caspase-1、IL-6、IL-1β、IL-18和TNF-α等炎症相关基因的转录水平,发现宿主在感染CIV早期炎症相关基因转录水平显著降低,而在感染H3N2 CIV后期的炎症相关基因转录水平比早期显著升高。进一步克隆犬NLRP3、ASC和Caspase-1基因并进行原核表达,制备多克隆抗体,通过免疫组织化学方法对未感染以及感染H3N2 CIV早期和后期的比格犬组织中的NLRP3、ASC(apoptosis-associated speck-like protein containing CARD)和Caspase-1蛋白的表达水平进行检测。结果发现在感染H3N2 CIV早期的犬组织中NLRP3、ASC和Caspase-1蛋白的阳性反应与对照组相比总体上没有明显的变化,而在感染后期犬组织中NLRP3、ASC和Caspase-1蛋白阳性反应明显增强。本研究为进一步研究流感病毒与宿主炎症反应的关系提供了思路,为后期更好地预防和治疗犬流感提供了理论依据。Inflammation is an innate immune response of the body against foreign bodies.NLRP3(nod-like receptor family,pyrin domain-containing 3 inflammasome pathway is one of the main pathways of inflammatory response,which plays an important role in the body?s response to pathogenic microorganisms.In order to study the changes of NLRP3 inflammasomes were after H3 N2 canine influenza virus infection in dogs,the heart,liver,spleen,and lungs of beagle dogs that are not infected and infected with H3 N2 CIV in the early(3 d)and late(7 d)phases,Kidney,brain,trachea and lymph node tissues were pathologically examined.The results showed that beagle dogs infected with H3 N2 CIV had no obvious pathological changes compared with the control group in the early stage,and the inflammatory response was not obvious,but the inflammatory response was significantly enhanced in the later stage.Secondly,fluorescence quantitative PCR was used to detect the transcription levels of inflammation-related genes such as NLRP3,ASC,Caspase-1,IL-6,IL-1β,IL-18and TNF-αafter H3N2CIV infection,and it was found that the host was infected with CIV.The transcription level of inflammation-related genes in the early stage was significantly reduced,while the transcription of inflammation-related genes in the late stage of H3N2CIV infection was significantly higher than that in the early stage.Moreover,we further cloned the canine NLRP3,ASC and Caspase-1genes and expressed them by prokaryotic expression.Polyclonal antibodies were prepared.Immunohistochemistry was used to detect NLRP3,NLRP3,ASC and Caspase-1in tissues of dogs uninfected and infected with H3N2CIV at early and late stages.The expression levels of ASC(apoptosis-associated speck-like protein containing CARD)and Caspase-1protein were detected.The results showed that the positive reaction of NLRP3,ASC and Caspase-1protein in the canine tissue at the early stage of infection with H3N2CIV did not change significantly compared with the control group,while the positive reaction of the NLRP3,ASC and Caspa

关 键 词：H3N2犬流感病毒 NLRP3炎症小体 NS1蛋白 

分 类 号：S852.65[农业科学—基础兽医学]