干扰素刺激基因ISG15对猪流行性腹泻病毒复制的作用及机制分析  

Effect and Mechanism of Interferon Stimulated Gene ISG 15 on Porcine Epidemic Diarrhea Virus Replication

作　　者：刘莉莉 边缘 吴圣龙[1] 包文斌[1] 吴正常[1] LIU Lili;BIAN Yuan;WU Shenglong;BAO Wenbin;WU Zhengchang(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《中国畜牧兽医》2024年第6期2545-2555,共11页China Animal Husbandry & Veterinary Medicine

基　　金：江苏省农业科技自主创新资金项目(CX(23)3084);省种业振兴“揭榜挂帅”项目(JBGS[2021]098)。

摘　　要：【目的】探究干扰素刺激基因ISG15在猪流行性腹泻病毒(PEDV)复制中所发挥的作用及机制。【方法】利用实时荧光定量PCR检测ISG15基因组织表达谱及肠道组织差异表达情况,同时以猪小肠上皮细胞(IPEC-J2)为研究模型,检测PEDV CV777毒株感染细胞不同时间点PEDV M基因mRNA和N蛋白表达量,并从RNA和蛋白水平检测ISG15表达变化;分别构建猪ISG15基因干扰和过表达细胞,通过实时荧光定量PCR、Western blotting及间接免疫荧光试验检测ISG15基因表达对PEDV复制水平的影响;对ISG15基因过表达前后进行转录组测序分析,筛选其下游调控基因及信号通路。【结果】ISG15基因在仔猪肠道组织中特异性高表达,其中空肠和回肠中表达量极显著高于其他组织(P<0.01);PEDV感染组十二指肠、空肠及回肠中ISG15基因表达量显著或极显著高于健康组(P<0.05;P<0.01);M基因mRNA和N蛋白表达量出现上升趋势,与0 h相比,ISG15基因表达水平在24 h出现极显著上调(P<0.01);ISG15基因过表达后PEDV复制出现显著或极显著下降(P<0.05;P<0.01),而ISG15基因干扰后PEDV复制出现极显著上调(P<0.01);转录组测序发现,过表达ISG15基因前后存在1 532个差异表达基因,且其主要富集在自噬、MAPK、内吞等信号通路中。【结论】本研究揭示了PEDV感染过程中ISG15基因的调控功能及作用机制,发现ISG15基因上调可显著抑制PEDV复制,增进了对PEDV与宿主细胞互作分子机制的认识。【Objective】The aim of this study was to investigate the role and mechanism of interferon-stimulated gene ISG 15 in the replication of Porcine epidemic diarrhea virus(PEDV).【Method】Real-time quantitative PCR was used to detect the tissue expression profile of ISG 15 gene and the differential expression in intestinal tissues.Meanwhile,porcine small intestine epithelial cells(IPEC-J2)were used as the study model to detect the expression of PEDV M gene mRNA and N protein of PEDV CV777 infected cells at different time points.The expression of ISG15 was detected at RNA and protein levels.Porcine ISG 15 gene interference and overexpression cells were constructed,and the influence of ISG 15 gene expression on PEDV replication level was detected by Real-time quantitative PCR,Western blotting and indirect immunofluorescence assay.Transcriptome sequencing was performed before and after the overexpression of ISG 15 gene to screen its downstream regulatory genes and signaling pathways.【Result】ISG 15 gene was specifically highly expressed in intestinal tissue of piglets,with significantly higher expression in jejunum and ileum compared with other tissues(P<0.01).The expression of ISG 15 gene in duodenum,jejunum and ileum of PEDV infected group was significantly or extremely significantly higher than that of healthy group(P<0.05 or P<0.01).The expression of PEDV M gene mRNA and N protein showed an upward trend,and compared with 0 h,the expression of ISG 15 gene was extremely significantly upregulated at 24 h(P<0.01).After overexpression of ISG 15 gene,there was a significant or extremely significant decrease in PEDV replication(P<0.05 or P<0.01),while after interference with ISG 15 gene,PEDV replication was extremely significantly upregulated(P<0.01).Transcriptome sequencing revealed 1532 differentially expressed genes before and after overexpression of the ISG 15 gene,which were mainly enriched in signaling pathways such as autophagy,MAPK signaling,and endocytosis.【Conclusion】This study revealed the regulatory

关 键 词：猪流行性腹泻病毒(PEDV) ISG15基因 病毒复制 转录组 

分 类 号：S852.651[农业科学—基础兽医学]