MID2对猪繁殖与呼吸综合征病毒复制的调控作用

doi:10.11843/j.issn.0366-6964.2023.11.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (11): 4735-4744.doi: 10.11843/j.issn.0366-6964.2023.11.027

MID2对猪繁殖与呼吸综合征病毒复制的调控作用

周立坤¹, 董鑫媛¹, 李家辉¹, 崔志莹², 赵士杰², 徐婕¹, 陈静^1*, 张宜娜^2*, 夏平安²

1. 河南农业大学生命科学学院, 郑州 450002;
2. 河南农业大学动物医学院, 郑州 450002

收稿日期:2023-02-28 出版日期:2023-11-23 发布日期:2023-11-26
通讯作者: 陈静,主要从事动物病毒与分子免疫学、动物病毒与宿主相互作用的机制研究,E-mail:chenjing@henau.edu.cn;张宜娜,主要从事分子病毒学与分子免疫学研究,E-mail:yinazhang2020@henau.edu.cn
作者简介:周立坤(2000-),男,河南信阳人,硕士生,主要从事病毒与分子免疫学的研究,E-mail:zlk2436@163.com
基金资助:
河南省重点研发与推广专项(科技攻关)项目(222102110297);国家青年科学基金资助项目(31802166)

Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication

ZHOU Likun¹, DONG Xinyuan¹, LI Jiahui¹, CUI Zhiying², ZHAO Shijie², XU Jie¹, CHEN Jing^1*, ZHANG Yina^2*, XIA Ping’an²

1. College of Life Science, Henan Agricultural University, Zhengzhou 450002, China;
2. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received:2023-02-28 Online:2023-11-23 Published:2023-11-26

摘要/Abstract

摘要： 本试验旨在研究MID2(midline 2)对猪繁殖与呼吸综合征病毒 (porcine reproductive and respiratory syndrome virus, PRRSV)复制的调控作用。首先用PRRSV BJ-4毒株感染MARC-145细胞,通过RT-qPCR和Western blot技术分别检测MID2的mRNA转录水平和蛋白表达水平,发现PRRSV感染能够上调MID2的表达。为了进一步探究MID2对PRRSV复制的影响,本研究过表达或沉默MID2表达,再接种PRRSV,通过RT-qPCR和Western blot技术,检测PRRSV ORF7的转录水平及N蛋白的表达水平,结果显示MID2能够抑制PRRSV复制。随后将pCMV-Flag-MID2和pGL3-IFN-β-Luc真核表达质粒共转染到HEK293T细胞中,通过双荧光素酶报告基因试验检测IFN-β的启动子活性,结果表明MID2能够正调控I-IFN信号通路。最后将MID2与RIG-I的真核表达质粒共转染到HEK293T细胞中,通过Co-IP试验发现MID2和RIG-I存在相互作用,且MID2能够以剂量依赖的方式促进RIG-I的表达,增强RIG-I介导的I-IFN信号通路。以上结果说明,MID2可能通过与RIG-I互作,促进RIG-I的表达,并激活I-IFN信号通路,从而抑制PRRSV复制。本研究首次发现MID2通过正调控I-IFN信号通路抑制PRRSV复制。

关键词: MID2, 猪繁殖与呼吸综合征病毒, I-IFN, RIG-I

Abstract: The purpose of this experiment was to study the regulatory effect of MID2 on porcine reproductive and respiratory syndrome virus (PRRSV) replication. Firstly, MARC-145 cells were infected with BJ-4 strain of PRRSV, and then the mRNA and protein expression level of MID2 were detected by RT-qPCR and Western blot. It was found that PRRSV infection could up-regulate MID2 expression. To further evaluate the effect of MID2 on PRRSV replication, this study overexpressed or knocked down the expression level of MID2 protein, and then inoculated PRRSV. It was found that MID2 could inhibit the replication of PRRSV by detecting the transcription level of PRRSV ORF7 and the expression level of N protein. Furthermore, the pCMV-Flag-MID2 and pGL3-IFN-β-Luc plasmid were co-transfected into HEK293T cells, the result of double luciferase assay showed that MID2 could positively regulate the type I interferon signaling pathway. Finally, the plasmid of MID2 and RIG-I were co-transfected into HEK293T cells, the result of Co-IP assay indicated that MID2 could interact with RIG-I, and MID2 could promote the protein expression level of RIG-I in a dose-dependent manner and enhance RIG-I mediated I-IFN signaling pathway. These results suggested that MID2 could activate the type I IFN signaling pathway through interacting with RIG-I and promoting RIG-I expression, lead to inhibiting PRRSV replication. In this study, we found for the first time that MID2 can positively regulate the type I interferon signaling pathway and inhibit PRRSV replication.

Key words: MID2, porcine reproductive and respiratory syndrome virus, I-IFN, RIG-I

中图分类号:

S852.659.6

周立坤, 董鑫媛, 李家辉, 崔志莹, 赵士杰, 徐婕, 陈静, 张宜娜, 夏平安. MID2对猪繁殖与呼吸综合征病毒复制的调控作用[J]. 畜牧兽医学报, 2023, 54(11): 4735-4744.

ZHOU Likun, DONG Xinyuan, LI Jiahui, CUI Zhiying, ZHAO Shijie, XU Jie, CHEN Jing, ZHANG Yina, XIA Ping’an. Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4735-4744.

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MID2对猪繁殖与呼吸综合征病毒复制的调控作用

Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication

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