摘要
为研究禽类疫病病原触发炎症的分子机制,试验克隆和分析鸡源白细胞介素-1β前体(Interleukin-1β precursors,pro-IL-1β)基因,原核表达和纯化pro-IL-1β蛋白,用纯化的pro-IL-1β蛋白免疫小鼠制备pro-IL-1β多克隆抗体,对该抗体进行ELISA效价检测和鉴定,并初步测试了该抗体在间接免疫荧光试验和Western blot试验中的应用效果。结果显示:重组蛋白pro-IL-1β成功表达,分子量约37 ku,能与His标签抗体发生特异性结合反应;获得了ELISA效价高于1∶32 000的鼠抗pro-IL-1β多克隆抗体,该抗体可通过间接免疫荧光试验(IFA)检测到pro-IL-1β真核表达蛋白,也可通过Western blot试验检测到pro-IL-1β原核表达蛋白和真核表达蛋白,还可通过Western blot和IFA检测到传染性法氏囊病病毒(IBDV)诱发的巨噬细胞HD11内源性pro-IL-1β蛋白的表达。研究表明,试验成功制备鸡pro-IL-1β多克隆抗体,为进一步研究禽病病原诱发炎症的分子机制提供了重要工具。
In order to study the molecular mechanism of inflammation triggered by avian infectious disease pathogens,the interleukin-1βprecursors(pro-IL-1β)gene from chicken was cloned and analyzed.The pro-IL-1βprotein was expressed and purified in prokaryotes.A pro-IL-1βpolyclonal antibody was prepared by immunizing mice with the purified pro-IL-1βprotein,and its ELISA titer was detected and identified.The application effect of the antibody in indirect immunofluorescence and Western blot was preliminarily tested.The results showed that the recombinant protein pro-IL-1βwas successfully expressed,with a molecular weight of about 37 ku,and could undergo specific binding reactions with His labeled antibodies;There was obtained mouse anti pro-IL-1βpolyclonal antibodies with ELISA titers higher than 1∶32000;This antibody could detect pro-IL-1βeukaryotic expression protein through indirect immunofluorescence assay(IFA),pro-IL-1βprokaryotic expression protein and eukaryotic expression protein through Western blot,and also detect the expression of endogenous pro-IL-1βprotein in macrophage HD11 induced by infectious bursal disease virus(IBDV)through Western blot and indirect immunofluorescence assay.The results indicated that preparation of chicken pro-IL-1βpolyclonal antibodies could provide important tools for further studying the molecular mechanisms of inflammation induced by avian pathogens.
作者
许萌萌
黄萌萌
牛鑫鑫
王国栋
于航博
张玉龙
韩京哲
韩金泽
刘润杭
吴子文
于晓雪
王笑梅
高玉龙
李留安
祁小乐
XU Mengmeng;HUANG Mengmeng;NIU Xinxin;WANG Guodong;YU Hangbo;ZHANG Yulong;HAN Jingzhe;HAN Jinze;LIU Runhang;WU Ziwen;YU Xiaoxue;WANG Xiaomei;GAO Yulong;LI Liu′an;QI Xiaole(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384;Avian Immunosuppressive Diseases Division,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin,Heilongjiang 150069;World Organization for Animal Health(WOAH)Reference Laboratory for Infectious Bursal Disease,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin,Heilongjiang 150069)
出处
《中国家禽》
北大核心
2024年第10期89-96,共8页
China Poultry
基金
国家自然科学基金项目(U20A2061、32072852)
“十四五”国家重点研发计划项目(2022YFD1800300)。
