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

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。

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.