猪源ISG15蛋白通过促进炎症因子释放和巨噬细胞死亡加剧胸膜肺炎放线杆菌感染  

Porcine ISG15 exacerbates Actinobacillus pleuropneumoniae infection by promoting inflammatory cytokine release and macrophage death

作　　者：甘林 文宇 田岩岩 张开心 李丰阳 雷连成 李娜 GAN Lin;WEN Yu;TIAN Yanyan;ZHANG Kaixin;LI Fengyang;LEI Liancheng;LI Na(Key Laboratory of Zoonosis Research,Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,Jilin,China)

机构地区：[1]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《微生物学通报》2025年第2期713-724,共12页Microbiology China

基　　金：国家自然科学基金(32273006)。

摘　　要：【背景】猪传染性胸膜肺炎是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)感染引起的一种呼吸道疾病,该病死亡率可达80%–100%。干扰素刺激基因15(interferon-stimulated genes 15,ISG15)蛋白可抑制病毒的复制,但其在APP方面的研究尚不清楚。【目的】构建猪源ISG15原核表达载体,纯化ISG15蛋白并研究其对APP感染的影响。【方法】构建pET-28a-ISG15原核表达载体,纯化获得ISG15蛋白。采用流式细胞术和实时荧光定量PCR法检测ISG15蛋白处理后肺泡巨噬细胞死亡情况和炎症因子表达水平。建立APP感染小鼠模型,鼻内给药ISG15后,检测小鼠体重、临床症状及存活情况,分析ISG15对APP致病性的影响。【结果】获得了ISG15蛋白。相较于单独攻菌组,ISG15添加组的肺泡巨噬细胞死亡率、炎症因子IL-6和IFN-γ表达水平均显著升高(P<0.05)。在小鼠试验中,相较于单独攻菌组,中剂量和高剂量ISG15组小鼠的临床症状及死亡率显著上升,肺组织IL-6、IFN-γ和IL-1β表达水平显著增加。【结论】ISG15通过促进肺泡巨噬细胞死亡和炎症因子的释放加剧APP的感染,为APP防治技术的研发提供了理论基础。[Background]Porcine transmissible pleuropneumonia is a respiratory disease caused by Actinobacillus pleuropneumoniae infection,and the mortality rate of the disease can reach 80%–100%.The ISG15 protein inhibits viral replication,but its study in Actinobacillus pleuropneumoniae is unknown.[Objective]To build a prokaryotic expression vector for porcine ISG15,purify the protein,and study its effect on Actinobacillus pleuropneumoniae(APP)infection.[Methods]A prokaryotic expression vector pET-28a-ISG15 was constructed for the expression of ISG15.Flow cytometry and fluorescence quantitative PCR were used to detect the death of alveolar macrophages and the expression of inflammatory cytokines after ISG15 treatment.A mouse model of APP infection was established.After intranasal administration of ISG15,the body weight,clinical symptoms,and survival of the mice were measured to analyze the influence of ISG15 on the pathogenicity of APP.[Results]ISG15 was obtained successfully after prokaryotic expression and purification.The death of alveolar macrophages and the expression levels of inflammatory cytokines interleukin-6(IL-6)and interferon-gamma(IFN-γ)were increased after ISG15 treatment(P<0.05).Compared with the APP challenge group,the mice treated with medium-and high-dose ISG15 post-infection showed severe clinical symptoms,increased mortality,and up-regulated expression levels of IL-6,IFN-γ,and IL-1βin the lung tissue.[Conclusion]ISG15 aggravates the APP infection by promoting the death of alveolar macrophages and the expression of inflammatory cytokines.The findings provide a theoretical basis for developing methods for the prevention and treatment of APP.

关 键 词：胸膜肺炎放线杆菌 ISG15 炎症因子 巨噬细胞 

分 类 号：S858.28[农业科学—临床兽医学]