非洲猪瘟病毒C717R蛋白诱导小鼠炎症应答

Inflammatory response induced by African swine fever virus C717R protein in mice

【目的】通过炎症应答系统筛选,发现非洲猪瘟病毒(African swine fever virus,ASFV)C717R蛋白可诱导炎症反应,本研究旨在首次鉴定C717R蛋白功能,通过构建C717R重组慢病毒并感染BALB/c小鼠,探究其对炎症应答产生的影响。【方法】通过炎症小体表达系统筛选出诱导炎症应答的C717R蛋白,并构建C717R重组慢病毒。利用C717R重组慢病毒感染小鼠,使C717R在小鼠组织中表达。经实时荧光定量、蛋白质免疫印迹等方法检测C717R慢病毒包装、蛋白表达以及促炎细胞因子TNF-α、IL-1β、IL-6、IFN-β的变化。【结果】C717R蛋白在小鼠组织中正常表达。酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测发现,表达C717R蛋白的小鼠,血清中促炎细胞因子TNF-α、IL-1β、IL-6、IFN-β及IFN-γ分泌水平显著升高。实时荧光定量检测表达C717R蛋白的小鼠组织证实,C717R、TNF-α、IL-1β、IL-6的mRNA转录水平显著上调;蛋白质免疫印迹证实,C717R可诱导小鼠不同组织caspase-1和IL-1β的成熟。组织病理切片结果显示,表达C717R蛋白的BALB/c小鼠和对照组和相比,肝脏、心脏、肺脏等器官炎性细胞浸润程度较对照组更严重。【结论】ASFV的C717R蛋白表达诱导BALB/c小鼠产生炎症应答,为鉴定和阐明C717R蛋白介导的促炎新机制提供了重要依据。

[Objective]By screening the inflammatory response system,we discovered that the African swine fever virus(ASFV)protein C717R induced an inflammatory response.This study aims to reveal the function of C717R protein by constructing recombinant C717R-lentivirus and observing the recombinant-induced inflammatory response in BALB/c mice.[Methods]C717R was screened out by the inflammasome expression system and used to construct the recombinant C717R-lentivirus.Mice were infected with the recombinant C717R-lentivirus to enable the expression of C717R in mouse tissue.Quantitative real-time PCR,western blotting,and enzyme-linked immunosorbent assay were employed to examine the package of C717R-lentivirus,protein expression,and the serum levels of pro-inflammatory cytokines including tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),IL-6,and interferon-beta(IFN-β),respectively.[Results]C717R protein was normally expressed in mouse tissue.The serum levels of TNF-α,IL-1β,IL-6,IFN-γ,and IFN-βwere elevated in the expression of C717R protein in mice.The mRNA levels of C717R,TNF-α,IL-1β,and IL-6 were significantly up-regulated in the expression of C717R protein in mice.Western blotting demonstrated that expression of C717R protein induced the caspase-1 and IL-1βin different tissues.The lesions in the liver,heart,lungs,and other organs of BALB/c mice expressing C717R protein were severer than those in the control mice.[Conclusion]The expression of C717R protein induced an inflammatory response in BALB/c mice.The findings provide a basis for deciphering the mechanism of C717R protein-mediated inflammation.