弓形虫TR与ROP18双基因缺失及对NF-κB-IL-12-ROS信号通路相关因子的影响

Double Deletion of TR and ROP18 Genes in Toxoplasma gondii and Effects on the Factors Related to NF-κB-IL-12-ROS Signaling Pathway

为探究弓形虫的TR与ROP18基因对NF-κB-IL-12-ROS信号通路的影响和抗宿主ROS损伤中是否具有协同作用,本研究利用CRISPR/Cas9技术构建了TR与ROP18双基因敲除株(TR-ROP18-KO)。通过检测TR/ROP18单、双基因缺失株氧化应激水平以及对小鼠巨噬细胞活性氧(ROS)水平影响的研究发现,TR-ROP18-KO株引发的氧化应激水平极显著高于RH株(P<0.01),但与TR单基因缺失株无显著差异(P>0.05);利用RT-PCR检测TR/ROP18单、双基因缺失株感染小鼠巨噬细胞产生的NF-κB、IL-12 mRNA水平和ELISA检测小鼠血清中IL-12的水平发现,TR-ROP18-KO株感染组虽然极显著高于RH株感染组(P<0.01),但TR-ROP18-KO株感染组却未显著高于TR-KO株感染组(P>0.05),表明弓形虫TR与ROP18基因在抗宿主ROS损伤中不存在协同作用。本研究利用TR与ROP18双基因缺失株能够明确解析这两个基因在抗宿主ROS损伤中有无协同作用,为深入研究这两个基因在I型弓形虫免疫逃避途径中的协同作用奠定基础,这也为后续弓形虫双基因功能协同作用的研究提供了一个新思路。

To investigate the effects of TR and ROP18 genes of Toxoplasma gondii on the NF-κB-IL-12-ROS signaling pathway and whether there is a synergistic effect of those two genes on resisting the ROS damage imposed by the host,In this study.A double gene deletion(TR-ROP18 KO)of TR and ROP18 was successfully constructed using CRISPR/Cas9 technology.By detecting the levels of oxidative stress in single and double deletion strains of TR/ROP18 and its effect on the level of reactive oxygen species(ROS)in mouse macrophages,it was found that the level of oxidative stress induced by TR-ROP18-KO strain was significantly higher than that of RH strain(P<0.01),but there was no significant difference between it and TR single-gene deletion strain(P>0.05).The mRNA levels of NF-κB and IL-12 produced by macrophages infected with TR/ROP18 single and double gene deletion strains were detected by RT-PCR,and serum levels of IL-12 were detected by ELISA.The results of the levels of NF-κB and IL-12 showed that the TR-ROP18-KO infection group was significantly higher than the RH infection group(P<0.01),but it was not significantly higher than the TR-KO infection group(P>0.05),indicating that TR and ROP18 genes have no synergistic effect on resisting the ROS damage from host.This study used TR and ROP18 double gene deletion strains to clearly resolve whether these two genes have a synergistic effect in resisting the ROS damage from the host,which may lay a foundation for in-depth study of the synergistic effect of these two genes of Toxoplasma type I on the immune escape pathway,and also provid a new idea for the subsequent study of the synergistic function of the double gene in Toxoplasma gondii.