Characteristics Analysis of TGEV Infection Mediated by IPEC-J2 with Knockout of pAPN Gene
The aim of this study was to explore the characteristics of porcine transmissible gastro-enteritis 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 inocula-tion 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％tis-sue culture infective dose(TCID50).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 TGEV did not affect the activity of the transcription factor NF-κB in the innate immunity-related signaling path-way in IPEC-J2-KO.This study provided the evidence that IPEC-J2 could serve as a cell model for the study of TGEV infection characteristics,and laid the foundation for elucidating the mech-anism of pAPN gene in TGEV invading host cells and researching new disease-resistant pig vari-eties.
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