单核细胞趋化蛋白诱导蛋白3对猪流行性腹泻病毒复制的体外抑制作用

doi:10.11843/j.issn.0366-6964.2025.06.028

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (6): 2847-2856.doi: 10.11843/j.issn.0366-6964.2025.06.028

单核细胞趋化蛋白诱导蛋白3对猪流行性腹泻病毒复制的体外抑制作用

周洪婷¹^,²(), 孙心如¹^,², 范宝超², 张雪寒², 周金柱², 索朗斯珠¹, 孙敏²^,*(), 贡嘎¹^,*(), 李彬²^,*()

1. 西藏农牧学院动物科学学院, 林芝 860019
2. 江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室, 南京 210014

收稿日期:2024-07-02 出版日期:2025-06-23 发布日期:2025-06-25
通讯作者: 孙敏,贡嘎,李彬 E-mail:3502582463@qq.com;sunmin9084@aliyun.com;xzlzgg@163.com;libinana@126.com
作者简介:周洪婷(1998-)，女，重庆垫江人，硕士生，主要从事高原动物传染病研究，E-mail：3502582463@qq.com
基金资助:
江苏省优秀青年基金项目(BK20230077);国家自然科学基金(32272996;32202787)

Inhibitory Effect of Monocyte Chemotactic Protein Induction Protein 3 on Porcine Epidemic Diarrhea Virus Replication in vitro

ZHOU Hongting¹^,²(), SUN Xinru¹^,², FAN Baochao², ZHANG Xuehan², ZHOU Jinzhu², SUOLANG Sizhu¹, SUN Min²^,*(), GONG Ga¹^,*(), LI Bin²^,*()

1. College of Animal Science, Xizang Agricultural and Animal Husbandry University, Nyingchi 860019, China
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China

Received:2024-07-02 Online:2025-06-23 Published:2025-06-25
Contact: SUN Min, GONG Ga, LI Bin E-mail:3502582463@qq.com;sunmin9084@aliyun.com;xzlzgg@163.com;libinana@126.com

摘要/Abstract

摘要：

旨在探究单核细胞趋化蛋白诱导蛋白3(monocyte chemotactic protein-induced protein 3，MCPIP3)对猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)复制的影响。通过RT-qPCR测定PEDV感染后ZC3H12C转录水平的动态变化；进而以猴肾上皮细胞cDNA为模板，PCR扩增出ZC3H12C基因，克隆到P3×Flag-CMV-14真核表达载体上，构建MCPIP3蛋白表达真核重组载体；分别于非洲绿猴胚胎肾细胞(MARC-145)上过表达或干扰MCPIP3蛋白，通过Western blot、荧光定量PCR、病毒滴度测定等方法探究其对PEDV复制的影响。结果表明，PEDV感染MARC-145细胞后显著上调ZC3H12C转录水平；成功构建MCPIP3重组质粒，其转染细胞后可诱导MCPIP3蛋白外源高表达，且特异性siRNA可显著下调ZC3H12C转录水平；与对照组相比，MCPIP3高表达后显著抑制PEDV N蛋白的表达水平，下调PEDV N基因的转录水平及细胞上清内病毒滴度，而干扰MCPIP3表达具有相反的效应，说明MCPIP3对PEDV在MARC-145细胞中的增殖具有显著抑制作用；同时，MCPIP3高表达显著下调PEDV感染诱导的IL-8、IL-12β及TNF-α的转录水平。本研究拓展了MCPIP3蛋白的功能研究，为PEDV复制机制研究提供了理论基础。

关键词: MCPIP3, PEDV, 病毒复制, 抗病毒作用

Abstract:

The purpose of this study was to investigate the effect of monocyte chemotactic protein-induced protein 3 (MCPIP3) on porcine epidemic diarrhea virus (PEDV) replication. The dynamic change of MCPIP3 transcription level after PEDV infection was determined by RT-qPCR. Then, using monkey kidney epithelial cell cDNA as template, ZC3H12C gene was amplified by PCR and cloned into P3×Flag-CMV-14 eukaryotic expression vector to construct MCPIP3 eukaryotic recombinant vector. MCPIP3 protein was overexpressed or interfered on African green monkey embryonic kidney cells (MARC-145), and the influence of MCPIP3 protein on PEDV replication was investigated by Western blot, quantitative PCR, and virus titer determination. The results showed that PEDV infection significantly up-regulated ZC3H12C transcription on MARC-145 cells. The MCPIP3 recombinant plasmid was successfully constructed, which could induce exogenous high expression of MCPIP3 protein after transfection, and the specific siRNA could significantly down-regulate the transcription level of ZC3H12C. Compared with the control group, high expression of MCPIP3 significantly inhibited PEDV N protein expression level, down-regulated PEDV N gene transcription levels and virus titer, while interfered with MCPIP3 expression had the opposite effect, indicating that MCPIP3 significantly inhibited PEDV proliferation on MARC-145 cells. Furthermore, the MCPIP3 expression negatively regulated PEDV induced transcription of IL-8, IL-12β and TNF-α. This study expanded the function of MCPIP3 protein, and provided a theoretical basis for the study of PEDV replication mechanism.

Key words: MCPIP3, PEDV, viral replication, antiviral effect

中图分类号:

S855.3

周洪婷, 孙心如, 范宝超, 张雪寒, 周金柱, 索朗斯珠, 孙敏, 贡嘎, 李彬. 单核细胞趋化蛋白诱导蛋白3对猪流行性腹泻病毒复制的体外抑制作用[J]. 畜牧兽医学报, 2025, 56(6): 2847-2856.

ZHOU Hongting, SUN Xinru, FAN Baochao, ZHANG Xuehan, ZHOU Jinzhu, SUOLANG Sizhu, SUN Min, GONG Ga, LI Bin. Inhibitory Effect of Monocyte Chemotactic Protein Induction Protein 3 on Porcine Epidemic Diarrhea Virus Replication in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2847-2856.

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基因 Gene	引物 Primer	序列(5'→3') Sequence
ZC3H12C	ZC3H12C-F	CGCCACCATGCCGGGTAGCGGCTCCCAGG
	ZC3H12C-R	ATAACCCAGCTGGGATTTCTCC
ZC3H12C-CHZ	ZC3H12C-CHZ-F	TGAACCGTCAGAATTAAGCTT CGCCACCATGCCGGGTAGCGGCTCCCAGG
	ZC3H12C-CHZ-R	TTTGTAGTCAGCCCGGGATCC ATAACCCAGCTGGGATTTCTCC

基因 Gene	干扰 RNA Interfering RNA	序列(5'→3') Sequence
ZC3H12C	si-MCPIP3-sense	CGUGCCAUGUCUAGAAAUATT
	si-MCPIP3-antisense	UAUUUCUAGACAUGGCACGTT
NC(Negative control)	NC-sense	UUCUCCGAACGUGUCACGUTT
	NC-antisense	ACGUGACACGUUCGGAGAATT

基因 Gene	引物 Primer	序列(5'→3') Sequence
m-GADPH	qm-GADPH-F	TGACCTGCCGTCTGGAAAA
	qm-GADPH-R	GGAAGAGTGGGTGTCGCTGT
m-ZC3H12C	qm-ZC3H12C-F	GAGAGTGGTGTGCTATGATGAC
	qm-ZC3H12C-R	CTCGTTAGCCAAATCCCTGTAG
m-IL-8	qm-IL-8-F	GACCACACTGCGTCAATACA
	qm-IL-8-R	AAACTTCTCCACAACCCTAGAC
m-IL-12β	qm-IL-12β-F	ACCAGAGTGGTGAGGTCTTA
	qm-IL-12β-R	CCTCCTTTGTGACAGGTGTATT
m-TNF-α	qm-TNF-α-F	AGCCCATGTTGTAGCAAACC
	qm-TNF-α-R	TGAGGTACAGGCCCTCTGAT
PEDV-N	PEDV-N-F	CGCAAAGACTGAACCCACTAACTT
	PEDV-N-R	TTGCCTCTGTTGTTACTCGGGGAT
	PEDV-N-probe	CTGTTGTTGCCATTGCCACGA

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单核细胞趋化蛋白诱导蛋白3对猪流行性腹泻病毒复制的体外抑制作用

Inhibitory Effect of Monocyte Chemotactic Protein Induction Protein 3 on Porcine Epidemic Diarrhea Virus Replication in vitro

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