鸡pro-IL-1β表达、多克隆抗体制备与初步应用  

Expression,Polyclonal Antibody Preparation and It′s Preliminary Application of Chicken Pro-IL-1β

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[2,3] 高玉龙[2,3] 李留安 祁小乐[2,3] 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)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室禽免疫抑制病创新团队,黑龙江哈尔滨150069 [3]中国农业科学院哈尔滨兽医研究所,世界动物卫生组织传染性法氏囊病参考实验室,黑龙江哈尔滨150069

出　　处：《中国家禽》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)。

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.

关 键 词：鸡 pro-IL-1β 克隆 表达 多克隆抗体 炎症 

分 类 号：S855.3[农业科学—临床兽医学]