稳定过表达NM-ⅡA Tail的IPEC-J2细胞系构建及其对PEDV感染的影响研究

doi:10.16431/j.cnki.1671-7236.2025.05.028

摘要/Abstract

摘要： 【目的】本研究利用慢病毒载体系统构建稳定过表达非肌性肌球蛋白ⅡA尾部(NM-ⅡA Tail)的猪小肠上皮细胞系(IPEC-J2)，探讨其在猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染细胞过程中的作用，为揭示PEDV的感染机制提供理论依据。【方法】采用同源重组技术构建过表达pLV-CMV-NM-ⅡA Tail-3×Flag-CopGFP-Puro的重组质粒。将重组质粒与辅助质粒pMD2.G和psPAX2共转染HEK-293FT细胞，制备慢病毒液。采用慢病毒液侵染IPEC-J2细胞，经药物筛选后观察EGFP荧光标签蛋白的表达情况；利用Western blotting和实时荧光定量PCR技术鉴定过表达NM-ⅡA Tail的细胞株构建情况。结合Western blotting和免疫荧光试验(IFA)分析PEDV内化阶段的变化，通过测定病毒粒子的拷贝量和病毒滴度，分析过表达NM-ⅡA Tail对PEDV复制和释放阶段的影响。【结果】在成功构建的过表达细胞株中，NM-ⅡA Tail mRNA转录水平较对照组提高了15倍(P＜0.01)，且Western blotting验证结果显示，NM-ⅡA Tail蛋白存在高效表达。在PEDV内化阶段，过表达NM-ⅡA Tail细胞中PEDV N蛋白表达量极显著高于对照组(P＜0.01)。IFA结果显示，过表达NM-ⅡA Tail细胞中NM-ⅡA和PEDV的荧光信号强于对照组。在PEDV复制阶段，过表达NM-ⅡA Tail细胞中PEDV M基因拷贝数及病毒半数组织感染剂量(TCID₅₀)显著或极显著高于对照组(P＜0.05；P＜0.01)。在PEDV释放阶段，过表达NM-ⅡA Tail细胞释放的PEDV M基因的拷贝数较对照组提高了2.7倍，上清中的病毒TCID₅₀极显著高于对照组(P＜0.01)。【结论】本研究成功构建了稳定过表达NM-ⅡA Tail的IPEC-J2细胞株，为深入研究NM-ⅡA在PEDV感染过程中的作用提供了试验材料。过表达NM-ⅡA Tail显著促进了PEDV的内化、复制和释放过程，为阐明PEDV的感染机制提供了新思路，并为疫病防控策略开发提供了参考依据。

关键词: 非肌性肌球蛋白ⅡA; 猪流行性腹泻病毒(PEDV); 慢病毒载体; 过表达细胞系

Abstract: 【Objective】 This study was aimed to construct a IPEC-J2 cell line stably overexpressing non-muscle myosin ⅡA tail (NM-ⅡA Tail) using a lentiviral vector system and investigate its role in the process of Porcine epidemic diarrhea virus (PEDV) infection,so as to provide a theoretical evidence for elucidating the infection mechanism of PEDV.【Method】 The recombinant plasmid pLV-CMV-NM-ⅡA Tail-3×Flag-CopGFP-Puro was constructed using homologous recombination technology.This recombinant plasmid was co-transfected with auxiliary plasmids pMD2.G and psPAX2 into HEK-293FT cells to produce the lentiviral particles.IPEC-J2 cells were infected with Lentivirus and subjected to drug selection,the expression of EGFP fluorescent tag protein was observed,and the construction of NM-ⅡA Tail-overexpressing cell line was confirmed using Western blotting and Real-time quantitative PCR.Western blotting and immunofluorescence assay (IFA) were used to examine the change during the PEDV internalization stage.The effects of NM-ⅡA Tail overexpression on PEDV replication and release were analyzed by measuring viral particle copy numbers and viral titers.【Result】 In the successfully constructed overexpression cell line,the mRNA transcription level of NM-ⅡA Tail was 15 times higher than that in control group (P＜0.01),and Western blotting results confirmed the efficient expression of NM-ⅡA Tail protein.During the PEDV internalization stage,the expression of PEDV N protein in NM-ⅡA Tail-overexpressing cells was extremely significantly higher than that in control group (P＜0.01).IFA results showed stronger fluorescence signals for NM-ⅡA and PEDV in NM-ⅡA Tail-overexpression group compared with control group.During the PEDV replication stage,the copy number of PEDV M gene and viral tissue culture infectious dose 50% (TCID₅₀) in NM-ⅡA Tail-overexpressing cells were significantly or extremely significantly higher than that in control group (P＜0.05 or P＜0.01).During the PEDV release stage,compared with control group,the copy number of PEDV M gene in supernatant of NM-ⅡA Tail-overexpressing cells was 2.7 times higher,and the viral TCID₅₀ was extremely significantly increased (P＜0.01).【Conclusion】 This study successfully constructed a stable IPEC-J2 cell line overexpressing NM-ⅡA Tail,so as to provide a valuable experimental model for further investigation of the role of NM-ⅡA in PEDV infection mechanism.The results demonstrated that NM-ⅡA Tail overexpression significantly promoted PEDV internalization,replication and release,offering new insights into the infection mechanism of PEDV and potential strategies for disease prevention and control.

Key words: non-muscle myosin ⅡA (NM-ⅡA); Porcine epidemic diarrhea virus (PEDV); lentiviral vector; overexpressing cell line

中图分类号:

S852.65⁺9.6

黄小久, 雷磊, 彭小烨, 王开心, 陈英仪, 王济贤, 王玉格, 段德勇, 杨毅, 王爱兵. 稳定过表达NM-ⅡA Tail的IPEC-J2细胞系构建及其对PEDV感染的影响研究[J]. 中国畜牧兽医, 2025, 52(5): 2243-2252.

HUANG Xiaojiu, LEI Lei, PENG Xiaoye, WANG Kaixin, CHEN Yingyi, WANG Jixian, WANG Yuge, DUAN Deyong, YANG Yi, WANG Aibing. Construction of a IPEC-J2 Cell Line Stably Overexpressing NM-ⅡA Tail and Its Effect on Porcine Epidemic Diarrhea Virus Infection[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2243-2252.

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