稳定表达人IFITM3的猪IPEC-J2细胞系的构建及其对PEDV增殖的影响

doi:10.11843/j.issn.0366-6964.2025.10.037

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (10): 5180-5189.doi: 10.11843/j.issn.0366-6964.2025.10.037

稳定表达人IFITM3的猪IPEC-J2细胞系的构建及其对PEDV增殖的影响

唐金梦¹(), 余澍楠¹, 袁一心¹, 马雨辰¹, 杜文娟¹^,², 于林洋¹^,*(), 李永涛¹^,²^,³^,*()

1. 河南农业大学动物医学院，郑州 450046
2. 国家动物免疫学国际联合研究中心，郑州 450046
3. 动物病原与生物安全教育部重点实验室，郑州 450046

收稿日期:2025-01-03 出版日期:2025-10-23 发布日期:2025-11-01
通讯作者: 于林洋,李永涛 E-mail:tjm19991126@163.com;linyangyuhenau@163.com;yongtaole@126.com
作者简介:唐金梦(1999-)，女，河南商丘人，硕士生，主要从事动物冠状病毒入侵机制与防控策略研究，E-mail: tjm19991126@163.com
基金资助:
国家自然科学基金面上项目(32172839)；国家留学基金公派高级研究学者、访问学者、博士后项目(201908410129)；河南省博士后科研启动基金(HN2022124)

Construction of Porcine IPEC-J2 Cell Line Stably Expressing Human IFITM3 and Its Effect on PEDV Proliferation

TANG Jinmeng¹(), YU Shunan¹, YUAN Yixin¹, MA Yuchen¹, DU Wenjuan¹^,², YU Linyang¹^,*(), LI Yongtao¹^,²^,³^,*()

1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
2. National International Joint Research Center for Animal Immunology, Zhengzhou 450046, China
3. Key Laboratory of Animal Pathogens and BioSafety, Ministry of Education, Zhengzhou 450046, China

Received:2025-01-03 Online:2025-10-23 Published:2025-11-01
Contact: YU Linyang, LI Yongtao E-mail:tjm19991126@163.com;linyangyuhenau@163.com;yongtaole@126.com

摘要/Abstract

摘要：

前期研究通过全基因组CRISPR文库筛选人肝癌细胞中调控猪流行性腹泻病毒(PEDV)感染的宿主因子，鉴定出人干扰素诱导跨膜蛋白3(IFITM3)参与PEDV感染进程，但其在猪源细胞中的功能尚未明确。本研究旨在构建稳定表达人IFITM3(hIFITM3)的猪肠上皮IPEC-J2细胞系，并系统评估其对PEDV复制的影响。首先，构建携带hIFITM3基因的重组慢病毒载体，与包装质粒pMD2.G/psPAX2共转染HEK293T细胞制备病毒颗粒。随后，通过慢病毒感染IPEC-J2细胞，经嘌呤霉素筛选和有限稀释法获得单克隆稳转细胞株(IPEC-J2^hIFITM3)。Western blot、间接免疫荧光法(IFA)及RT-qPCR验证显示hIFITM3在靶细胞中高效表达。CCK-8试验证实hIFITM3过表达未显著影响细胞活力。功能验证表明，IPEC-J2^hIFITM3细胞可有效抑制水泡性口炎病毒(VSV-GFP)的早期复制，与其已知抗病毒特性一致。然而，感染PEDV DR13-GFP毒株后，IPEC-J2^hIFITM3细胞表现出显著的促病毒增殖效应：24 h时病毒滴度(TCID₅₀·mL^-1)较野生型细胞提高约1 000倍。本研究成功建立hIFITM3稳转猪肠上皮细胞模型，其反常促进PEDV复制的特性为解析病毒种属特异性感染机制、开发抗病毒策略及优化病毒分离培养体系提供了重要工具。

关键词: 猪流行性腹泻病毒, IPEC-J2细胞, 干扰素诱导跨膜蛋白3, 细胞系

Abstract:

Previous genome-wide CRISPR screening in human hepatocellular carcinoma cells identified human interferon-induced transmembrane protein 3 (IFITM3) as a critical host factor regulating porcine epidemic diarrhea virus (PEDV) infection. However, its functional role in porcine-derived cells remains unexplored. Here, we generated a porcine IPEC-J2 cell line stablely overexpressing hIFITM3 (IPEC-J2^hIFITM3) and systematically evaluated its impact on viral proliferation. The recombinant lentiviral vector encoding hIFITM3 was co-transfected with packaging plasmids pMD2.G/psPAX2 into HEK293T cells to produce replication-incompetent lentiviral particles. Following transduction of IPEC-J2 cells and puromycin selection, monoclonal cells were established via limiting dilution. Robust hIFITM3 overexpression was confirmed by Western blot, indirect immunofluorescence assay (IFA), and RT-qPCR. CCK-8 assays verified no significant cytotoxicity associated with hIFITM3 expression. Functional validation demonstrated that IPEC-J2^hIFITM3 cells potently suppressed vesicular stomatitis virus (VSV-GFP) replication, consistent with IFITM3's canonical antiviral function. Strikingly, challenge with PEDV DR13-GFP revealed a paradoxical pro-viral effect: IPEC-J2^hIFITM3 cells exhibited a 1 000-fold increase in viral titer (TCID₅₀ ·mL^-1) compared to wild-type cells at 24 hpi. This study establishes a porcine intestinal epithelial model stably expressing hIFITM3, which unexpectedly potentiates PEDV replication. These findings not only highlight the species-specific functional divergence of IFITM3 but also provide a unique platform for investigating PEDV-host interactions, optimizing viral cultivation, and developing targeted antiviral therapeutics.

Key words: porcine epidemic diarrhea virus, IPEC-J2 cells, IFITM3, cell line

中图分类号:

S852.659.6

唐金梦, 余澍楠, 袁一心, 马雨辰, 杜文娟, 于林洋, 李永涛. 稳定表达人IFITM3的猪IPEC-J2细胞系的构建及其对PEDV增殖的影响[J]. 畜牧兽医学报, 2025, 56(10): 5180-5189.

TANG Jinmeng, YU Shunan, YUAN Yixin, MA Yuchen, DU Wenjuan, YU Linyang, LI Yongtao. Construction of Porcine IPEC-J2 Cell Line Stably Expressing Human IFITM3 and Its Effect on PEDV Proliferation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5180-5189.

图/表 7

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参考文献 26

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引物名称 Primer name	引物序列 Primer sequence
Sus-GAPDH：F(5′→3′)	TCGGAGTGAACGGATTTGGC
Sus-GAPDH：R(5′ →3′)	TGCCGTGGGTGGAATCATAC
Sus-IFITM3：F(5′ →3′)	TGCTCTAGAATGAATTGCGCTTCCCAGCC
Sus-IFITM3：R(5′ →3′)	CGGGATCCTTAGTGGTGGTGGTGGTGGTGCTAGTAG
Hu-IFITM3：F(5′ →3′)	TCACACTGTCCAAACCTTCTTC
Hu-IFITM3：R(5′ →3′)	GGATGTGGATCACGGTGGAC
PQCXIP-Flag-hIFITM3质粒：F(5′)	ACGCCATCCACGCTGTTTTGACCT
PQCXIP-Flag-hIFITM3质粒：R(3′)	AAGCGGCTTCGGCCAGTAACGTTA

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稳定表达人IFITM3的猪IPEC-J2细胞系的构建及其对PEDV增殖的影响

Construction of Porcine IPEC-J2 Cell Line Stably Expressing Human IFITM3 and Its Effect on PEDV Proliferation

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