猪流行性腹泻病毒感染对猪小肠上皮细胞I型干扰素产生通路的影响  

Effects of porcine epidemic diarrhea virus infection on IFN-βproduction pathway in porcine intestinal epithelial cells

作　　者：丁芳艺 宋海鑫 梁荣 苗晋锋[2] 费荣梅[2] 刘永杰[2] 余祖功[2] 张金秋[1,3,4,5] DING Fang-yi;SONG Hai-xin;LIANG Rong;MIAO Jin-feng;FEI Rong-mei;LIU Yong-jie;YU Zu-gong;ZHANG Jin-qiu(Institute of Veterinary Immunology and Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;Jiangsu Key Laboratory for Food Quality and Safety——State Key Laboratory Cultivation Base, Nanjing 210014, China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;College of Pharmacy, Jiangsu University, Zhenjiang 212013, China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]省部共建国家重点实验室培育基地——江苏省食品质量安全重点实验室,江苏南京210014 [4]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [5]江苏大学药学院,江苏镇江212013

出　　处：《江苏农业学报》2021年第1期99-105,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31772701);江苏省农业科技自主创新基金项目[CX(17)3027]。

摘　　要：猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是严重危害仔猪肠道健康的病原之一。用病毒感染复数(MOI)=1.0的PEDV感染猪小肠上皮细胞,通过间接免疫荧光试验,证实PEDV能在猪小肠上皮细胞中增殖。通过实时荧光定量PCR(RT-qPCR)检测,发现Toll样受体3(TLR3)编码基因、视黄醇诱导基因I/黑色素瘤分化相关基因5(RIG-I/MDA5)的mRNA表达水平分别从感染后2 h、4 h开始显著升高(P<0.05),分别在感染后12 h、24 h达到最高值;线粒体抗病毒信号蛋白(MAVS)编码基因的mRNA相对表达量从感染后6 h开始显著升高(P<0.05),随后基本保持不变。下游接头蛋白质β干扰素TIR结构域衔接蛋白质(TRIF)、肿瘤坏死因子相关因子3(TRAF3)、IκB激酶ε(IKK-ε)和TANK结合激酶1(TBK1)编码基因的mRNA相对表达量在感染后4 h出现显著上升(P<0.05),随后IKK-ε编码基因的mRNA相对表达量在感染后8 h达到最高值,TRIF、TRAF3和TBK1编码基因的mRNA相对表达量均在感染后12 h达到最高值。干扰素调节因子3(IRF3)编码基因的mRNA相对表达量在感染早期无显著变化,在感染后24 h达到最高值(P<0.05)。与对照组相比,PEDV感染无法诱导猪小肠上皮细胞(IPEC-J2)IFN-β编码基因的mRNA相对表达量显著升高,但能有效抑制Poly(I:C)(聚肌胞苷酸)诱导的IFN-β产生。结果表明,PEDV能够在感染早期激活TLRs、RLRs介导的I型IFN产生通路中相关受体及接头蛋白质的基因表达,但最终能抑制干扰素产生,并能在猪小肠上皮细胞中有效增殖。Porcine epidemic diarrhea virus(PEDV)is one of the pathogens that seriously endanger the intestinal health of piglets.Porcine intestinal epithelial cells(IPEC-J2)were infected with PEDV at a multiplicity of infection MOI of 1.0.Virus proliferation in IPEC-J2 was observed by indirect immunofluorescence assay(IFA).Through real-time fluorescent quantitative PCR(RT-PCR)detection,it was found that the mRNA levels of TLR3 encoding gene and RIG-I/MDA5 significantly increased from 2 hours post infection(hpi)and 4 hpi,respectively(P<0.05),and peaked at 12 hpi and 24 hpi.The relative mRNA expression of mitochondrial antiviral signaling protein(MAVS)coding gene significantly increased at 6 hpi(P<0.05),and then remained unchanged.The mRNA levels of TRIF,TRAF3,IKK-εand TBK1 coding genes significantly increased from 4 hpi(P<0.05),and then the relative mRNA expression of IKK-εencoding genes reached the highest level at 8 hpi while TRIF,TRAF3 and TBK1 encoding genes at 24 hpi.The relative mRNA expression of interferon regulatory factor 3(IRF3)encoding gene showed no significant change at the early stage after infection,and reached the highest level at 12 hpi.Compared with control group,there was no significant difference of IFN mRNA expression in IPEC-J2 infected with PEDV.However,PEDV infection could inhibit the production of IFN-βinduced by Poly(I:C).The results suggested that PEDV could activate TLRs and RLRs signaling pathway in the early stage after infection,and effectively proliferate in IPEC-J2 by inhibiting IFN-β.

关 键 词：天然免疫 猪流行性腹泻病毒 猪小肠上皮细胞 I型干扰素 

分 类 号：S828.7[农业科学—畜牧学]