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

Abstract

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

CLC Number:

S852.65⁺9.6

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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Construction of a IPEC-J2 Cell Line Stably Overexpressing NM-ⅡA Tail and Its Effect on Porcine Epidemic Diarrhea Virus Infection

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