猪繁殖与呼吸综合征病毒RNA刺激下NLRP3与DDX19A共定位的研究
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摘要
NLRP3蛋白为核苷酸结合寡聚化结构域(NOD)样受体(NLRs)家族中含有脓素(Pyrin)结构域3的蛋白,它能够与调亡相关斑点样蛋白(ASC)以及半胱氨酸蛋白水解酶1(Caspase-1)组成炎性小体复合物,切割pro-IL-1β和pro-IL-18使其成为成熟的IL-1β和IL-18参与炎性反应。猪繁殖与呼吸障碍综合征病毒(PRRSV)的RNA能够被DDX19A解旋酶识别并激活NLRP3炎性小体,诱导炎症反应。为研究PRRSV RNA激活NLRP3炎症小体后DDX19A与NLRP3的亚细胞定位情况,本研究以PRRSV Hu N4株感染猪肺泡巨噬细胞(PAMs)的总RNA制备的c DNA为模板,扩增了猪nlrp3基因。将其在原核细胞中表达,并采用表达的NLRP3免疫BALB/c小鼠制备了鼠源抗NLRP3多克隆抗体。此外,提取PRRSV的RNA,转染于PAMs中,36 h后分别采用抗DDX19A和抗NLRP3的多克隆抗体进行检测,激光共聚焦试验结果显示:未转染PRRSV RNA的细胞中,DDX19A和NLRP3在PAMs中呈弥散表达,无共定位现象出现,而在转染PRRSV RNA的PAMs中DDX19A和NLRP3在细胞质中呈点状分布并出现共定位现象。本研究为阐明PRRSV感染激活NLRP3炎性小体的分子机制奠定了基础。
NLRP3 is NOD-like receptor pyrin domain-containing protein 3 and the NLRP3 inflammasome which consists of NLRP3, apoptosis-associated speck like protein containing a caspase activation and recruitment domain(ASC), and caspase-1plays a major role in innate immune responses by activating caspase-1, resulting in secretion of IL-1β and IL-18. DDX19 A has been proved to sense porcine reproductive and respiratory syndrome virus(PRRSV) RNA and activate NLRP3 inflammasome. To study the function of PRRSV RNA in activating NLRP3 inflammasome and explore the localization of NLRP3 and DDX19 A after viral RNA stimulation, the nlrp3 gene was amplified from PRRSV infected porcine alveolar macrophage(PAMs) and expressed in E.coli. The polyclonal antibodies against NLRP3 were prepared in BALB/c mice immunized with the recombinant protein.Furthermore, PRRSV RNA was transfected into PAMs. The transfected PAMs were detected with anti-DDX19 A and anti-NLRP3 polyclonal antibodies. The confocal examination result showed that DDX19 A and NLRP3 were diffusedly expressed in cytoplasm of the non-transfected PAMs, whereas the co-localization of DDX19 A and NLRP3 were observed in PRRSV RNA transfectedPAMs. In conclusion, this result laid a foundation for further study on NLRP3 inflammasome to trigger the inflammatory responses for antivirus infection.
NLRP3 is NOD-like receptor pyrin domain-containing protein 3 and the NLRP3 inflammasome which consists of NLRP3, apoptosis-associated speck like protein containing a caspase activation and recruitment domain(ASC), and caspase-1plays a major role in innate immune responses by activating caspase-1, resulting in secretion of IL-1β and IL-18. DDX19 A has been proved to sense porcine reproductive and respiratory syndrome virus(PRRSV) RNA and activate NLRP3 inflammasome. To study the function of PRRSV RNA in activating NLRP3 inflammasome and explore the localization of NLRP3 and DDX19 A after viral RNA stimulation, the nlrp3 gene was amplified from PRRSV infected porcine alveolar macrophage(PAMs) and expressed in E.coli. The polyclonal antibodies against NLRP3 were prepared in BALB/c mice immunized with the recombinant protein.Furthermore, PRRSV RNA was transfected into PAMs. The transfected PAMs were detected with anti-DDX19 A and anti-NLRP3 polyclonal antibodies. The confocal examination result showed that DDX19 A and NLRP3 were diffusedly expressed in cytoplasm of the non-transfected PAMs, whereas the co-localization of DDX19 A and NLRP3 were observed in PRRSV RNA transfectedPAMs. In conclusion, this result laid a foundation for further study on NLRP3 inflammasome to trigger the inflammatory responses for antivirus infection.
引文
[1]Bi Jin g,Song Shuang,Fang Liu-rong,et al.Porcine reproductive and respiratory syndrome virus induces IL-1beta production depending on TLR4/MyD 88 pathway and NLRP3 inflammasome in primary porcine alveolar macrophages[J].Mediators Inflamm,2014,2014:403515.
[2]Li Jiang-nan,Hu Liang,Liu Yuan-yuan,et al.DDX19A senses viral RNA and mediates NLRP3-dependent inflammasome activation[J].J Immunol,2015,195(12):5732-5749.
[3]刘园园,李江南,刘琴芳,等.猪caspase-1 p20多克隆抗体制备及其在猪繁殖与呼吸障碍综合征病毒感染中的初步应用[J].中国预防兽医学报,2014,2:98-101.
[4]尹曼曼,郭东伟,刘琴芳,等.猪繁殖与呼吸综合征病毒N蛋白与BCCIP蛋白相互作用的鉴定[J].中国预防兽医学报,2013,5:341-345.
[2]Li Jiang-nan,Hu Liang,Liu Yuan-yuan,et al.DDX19A senses viral RNA and mediates NLRP3-dependent inflammasome activation[J].J Immunol,2015,195(12):5732-5749.
[3]刘园园,李江南,刘琴芳,等.猪caspase-1 p20多克隆抗体制备及其在猪繁殖与呼吸障碍综合征病毒感染中的初步应用[J].中国预防兽医学报,2014,2:98-101.
[4]尹曼曼,郭东伟,刘琴芳,等.猪繁殖与呼吸综合征病毒N蛋白与BCCIP蛋白相互作用的鉴定[J].中国预防兽医学报,2013,5:341-345.