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

摘要/Abstract

摘要： 【目的】探究长链非编码RNA(lncRNA)在猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)感染猪小肠上皮细胞(IPEC-J2)过程中的作用。【方法】利用感染复数(MOI)为1的PEDV感染IPEC-J2细胞构建细胞病变模型，通过RNA-Seq进行lncRNA测序，筛选出影响PEDV复制的关键lncRNA；利用实时荧光定量PCR和核质分离检测lncRNA表达和细胞定位；构建RNA干扰载体并转染IPEC-J2细胞，通过病毒拷贝数测定、Western blotting、半数组织培养感染剂量(TCID₅₀)以及间接免疫荧光试验(IFA)检测lncRNA表达对PEDV复制水平的影响。【结果】 PEDV感染IPEC-J2细胞24 h时，细胞病变最为明显，成功构建细胞病变模型。与对照组相比，PEDV感染组中存在61个差异表达lncRNA，其中19个上调，42个下调，筛选出1个显著上调且差异倍数较大的lncRNA——lncMSTRG.10733.2。实时荧光定量PCR结果显示，与对照组相比，PEDV感染IPEC-J2细胞后，lncMSTRG.10733.2表达水平显著上调(P＜0.05)。核质分离测定结果显示，lncMSTRG.10733.2主要分布在IPEC-J2细胞质中。成功构建了lncMSTRG.10733.2瞬时干扰IPEC-J2细胞，干扰效率为45.9%。实时荧光定量PCR和Western blotting结果显示，lncRNA干扰后，PEDV-M基因mRNA相对表达量和PEDV N蛋白表达水平均显著升高(P＜0.05)。TCID₅₀测定结果显示，lncRNA干扰后PEDV滴度极显著上升(P＜0.01)；IFA结果显示，lncRNA干扰后IPEC-J2细胞中的PEDV N蛋白荧光分布水平明显增加。【结论】本研究基于高通量测序从细胞水平上筛选鉴定出1个与PEDV感染密切相关的lncRNA，其表达水平上调有利于提高猪对PEDV感染的抵抗能力。研究结果揭示了lncRNA在PEDV感染宿主过程中的重要作用，为今后制定PEDV的抗病育种工作策略和筛选分子标记物奠定基础。

关键词: 猪流行性腹泻病毒(PEDV); lncRNA; IPEC-J2; 病毒复制

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

中图分类号:

S852.65⁺1

杨荔, 杜小妹, 刘梦媛, 吴圣龙, 包文斌, 吴正常. 影响猪流行性腹泻病毒复制的关键lncRNA筛选及其功能验证[J]. 中国畜牧兽医, 2025, 52(2): 792-800.

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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