pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析  

Characteristics Analysis of TGEV Infection Mediated by IPEC-J2 with Knockout of pAPN Gene

作　　者：夏振涛 王楠[1] 王婉洁 周期律 黄雷 牟玉莲[1] XIA Zhentao;WANG Nan;WANG Wanjie;ZHOU Qilü;HUANG Lei;MU Yulian(Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业科学院农业基因组研究所,深圳518120

出　　处：《畜牧兽医学报》2024年第8期3395-3407,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：深圳市科技计划项目(CJGJZD20210408092402006);中国农业科学院科技创新工程(ASTIP-IAS05)。

摘　　要：旨在探究猪氨基肽酶N(porcine aminopeptidase N,pAPN)基因敲除的猪空肠上皮细胞系(intestinal porcine epithelial cell line J2,IPEC-J2)介导猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)感染的特征,为深入了解pAPN基因在TGEV感染过程中的作用机制提供理论依据。研究分为pAPN基因敲除IPEC-J2组(IPEC-J2-KO组)、野生型IPEC-J2组(IPEC-J2-WT组)和未接种TGEV的野生型IPEC-J2组(Mock组)3组,每组设置3个重复。首先通过实时荧光定量PCR(quantitative real-time PCR,qPCR)确定IPEC-J2接种TGEV毒株后收取细胞样品的最佳时间节点;其次,对IPEC-J2-KO进行了脱靶效应检测;然后,通过qPCR、蛋白免疫印迹(western blot,WB)、间接免疫荧光分析(indirect immunofluorescence assay,IFA)和50%组织细胞感染量(50%tissue culture infective dose,TCID_(50))对接种TGEV的IPEC-J2-KO、IPEC-J2-WT及Mock进行感染特征分析;最后,通过WB检测IPEC-J2-KO、IPEC-J2-WT和Mock中NF-κB p65及其磷酸化蛋白pp65的表达情况。qPCR结果显示,接毒24 h是收集细胞样本以评估TGEV对IPEC-J2影响的最佳时间节点;脱靶分析结果显示,在IPEC-J2-KO中未检测到脱靶效应;病毒感染特征分析结果显示,与IPEC-J2-WT相比,IPEC-J2-KO内病毒拷贝数、病毒滴度均极显著降低(P<0.001),与Mock相比,IPEC-J2-KO内病毒拷贝数、病毒滴度均无显著差异(P>0.05),且IPEC-J2-KO内未检测到TGEV-N蛋白的表达;此外,与Mock相比,接种TGEV后,IPEC-J2-WT组NF-κB p65的磷酸化水平极显著上调(P<0.001),而IPEC-J2-KO组无显著差异(P>0.05)。本研究表明,IPEC-J2-KO可有效抵抗TGEV的感染,接种TGEV未影响IPEC-J2-KO中先天免疫相关信号通路中转录因子NF-κB的活性。该研究为IPEC-J2作为TGEV感染特征研究的细胞模型提供了理论依据,为阐明pAPN基因在TGEV入侵宿主细胞的机制及抗病猪新品种的研究奠定了基础。The aim of this study was to explore the characteristics of porcine transmissible gastroenteritis virus(TGEV)infection mediated by porcine aminopeptidase N(pAPN)gene knockout intestinal porcine epithelial cell line J2(IPEC-J2)and to provide theoretical basis for further understanding the mechanism of pAPN gene in the process of TGEV infection.The study was divided into 3 groups,pAPN gene knockout IPEC-J2 group(IPEC-J2-KO group),wild-type IPEC-J2 group(IPEC-J2-WT group),and wild-type IPEC-J2 group not inoculated with TGEV(Mock group),with 3 replicates set up in each group.Firstly,quantitative real-time PCR(qPCR)was used to determine the best time point for collecting cell samples following inoculation with TGEV strain.Secondly,the off-target effect of IPEC-J2-KO was detected.Then,the infection characteristics of IPEC-J2-KO,IPEC-J2-WT inoculated with TGEV and Mock were analyzed by qPCR,western blot(WB),indirect immunofluorescence assay(IFA),and 50%tissue culture infective dose(TCID _(50)).Finally,the expression of NF-κB p65 and its phosphorylated protein pp65 in Mock,IPEC-J2-WT and IPEC-J2-KO were detected by WB.The results of qPCR showed that 24 hours after exposure was the best time point to collect cell samples to evaluate the impact of TGEV on IPEC-J2;The off-target analysis results showed that no off-target effects were detected in IPEC-J2-KO;The results of virus infection characteristic analysis showed that the virus copy number and virus titer in IPEC-J2-KO were significantly lower than those in IPEC-J2-WT(P<0.001).Compared with the Mock,there was no significant difference in virus copy number and virus titer in IPEC-J2-KO(P>0.05),and no expression of TGEV-N protein was detected in IPEC-J2-KO.In addition,compared with the Mock,after inoculation with TGEV,the phosphorylation level of NF-κB p65 was significantly increased in the IPEC-J2-WT group(P<0.001),while there was no significant difference in IPEC-J2-KO group(P>0.05).This study results showed that IPEC-J2-KO can effectively resist TGEV infection,and TG

关 键 词：猪氨基肽酶N IPEC-J2 TGEV NF-ΚB 

分 类 号：S813.1[农业科学—畜牧学]