影响猪流行性腹泻病毒复制的关键lncRNA筛选及其功能验证

doi:10.16431/j.cnki.1671-7236.2025.02.029

Abstract

Abstract: 【Objective】 The experiment was to investigate the role of long non-coding RNA (lncRNA) in the infection of Porcine epidemic diarrhea virus (PEDV) in porcine small intestine epithelial cells (IPEC-J2). 【Method】 The cytopathic model was constructed using PEDV-infected IPEC-J2 cells with a multiplicity of infection (MOI) of 1.lncRNA sequencing was performed by RNA-Seq to screen out key lncRNA affecting PEDV replication.lncRNA expression and cell localization were detected by Real-time quantitative PCR and karyoplasmic separation.RNA interference vector was constructed and transfected with IPEC-J2 cells，then the effect of lncRNA expression on PEDV replication level was detected by virus copy number assay,Western blotting,50% tissue culture infectious dose (TCID₅₀) and indirect immunofluorescence assay (IFA). 【Result】 When PEDV infected IPEC-J2 cells for 24 h,the cytopathosis was most obvious,and the cytopathosis model was successfully constructed.Compared with control group,61 lncRNAs were differentially expressed in the PEDV infected group,among which 19 were up-regulated and 42 were down-regulated.lncRNA with significantly up-regulated and difference multiple was screened out—lncMSTRG.10733.2.Real-time quantitative PCR results showed that the expression of lncMSTRG.10733.2 was significantly up-regulated after PEDV infection with IPEC-J2 cells compared with control group (P＜0.05).lncMSTRG.10733.2 was mainly distributed in the cytoplasm of IPEC-J2 cells.lncMSTRG.10733.2 transient interference IPEC-J2 cells were successfully constructed,and the interference efficiency was 45.9%.Real-time quantitative PCR and Western blotting results showed that the relative mRNA expression level of PEDV-M gene and PEDV N protein expression level were significantly increased after lncRNA interference (P＜0.05).The results of TCID₅₀ showed that the virus titer of PEDV increased extremely significantly after lncRNA interference (P＜0.01).IFA results showed that the fluorescence distribution of PEDV N protein in IPEC-J2 cells was significantly increased after lncRNA interference. 【Conclusion】 In this study,one lncRNA closely related to PEDV infection was screened and identified at the cellular level based on high-throughput sequencing，and its up-regulated expression level was conducive to improving the resistance of pigs to PEDV infection.This study revealed the important role of lncRNA in the process of PEDV infection in the host,which laid a foundation for the development of PEDV resistance breeding strategies and the screening of molecular markers of PEDV in the future.

Key words: Porcine epidemic diarrhea virus (PEDV); lncRNA; IPEC-J2; viral replication

CLC Number:

S852.65⁺1

YANG Li, DU Xiaomei, LIU Mengyuan, WU Shenglong, BAO Wenbin, WU Zhengchang. Screening and Functional Verification of Key lncRNA Affecting Porcine Epidemic Diarrhea Virus Replication[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 792-800.

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Screening and Functional Verification of Key lncRNA Affecting Porcine Epidemic Diarrhea Virus Replication

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