鸡组织中禽波氏杆菌间接免疫荧光组化定位检测方法的建立  

Establishment of Indirect Fluorescent Immunohistochemistry for Localization Detection of Bordetella aviumin Chick Tissues

作　　者：杨萍萍[1] 赵雪[1] 刘静静[1] 贺晓华[1] 朱瑞良[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271000

出　　处：《中国畜牧兽医》2015年第11期3087-3092,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31272595;30972183);山东省自然科学基金项目(ZR2014CM044)

摘　　要：为研究禽波氏杆菌感染鸡后在体内组织器官中的定植规律及其在不同组织细胞中的定位、定量状况,本试验对禽波氏杆菌感染鸡组织器官的固定剂进行了筛选,制备石蜡切片,选择良好的粘片剂,利用纯化的兔抗禽波氏杆菌外膜蛋白IgG作为一抗,建立了能进行禽波氏杆菌抗原定位检测的间接免疫荧光组化法,并对禽波氏杆菌人工感染鸡进行了检测。经多次优化,确定将10%中性福尔马林作为固定剂固定组织24h,0.1%多聚-L-赖氨酸作为切片的粘片剂。最佳工作条件为2%脱脂奶粉37℃封闭30min;兔抗禽波氏杆菌外膜蛋白IgG 1∶100稀释,4℃孵育过夜;荧光素标记羊抗兔二抗1∶20稀释,37℃作用30min。该方法重复性和特异性检测良好,对禽波氏杆菌感染雏鸡组织器官的检测结果表明,该法能特异性地对鸡组织器官中的禽波氏杆菌进行抗原定位,气管、肺脏、肝脏、心脏、脾脏、肾脏中的禽波氏杆菌抗原均呈现特异性荧光信号,同细菌分离鉴定方法检测的阳性符合率为99.27%。试验结果表明,本试验所建立的间接免疫荧光组化法可作为一种有效的检测方法用于禽波氏杆菌致病机制的研究。In order to study the colonization pattern,localization and quantity of Bordetella avium（B.avium）in the tissues and organs of chicks infected with B.avium,a method of indirect fluorescent immunohistochemistry suitable for B.avium detection was established and applied to detect tissues of artificially infected chicks in this study.Firstly the fixation agent for fixing tissues and organs of chicks was screened.Then the fixed tissues were sliced to sections and good pasting agent was detected.Rabbit anti-B.aviumouter membrane protein IgG was used as the first antibody.After repeatedly optimizing the experiment conditions,10% neutral formalin was used as a fixation agent to fix tissues for 24 hand 0.1% poly-L-lysine as a section pasting agent.Optimal working conditions were as follows：2%skimmed milk powder closing section at 37℃for 30min;1∶100dilution of rabbit anti-B.aviumouter membrane protein IgG incubating at 4℃ overnight;Fluorescein marked goat anti-rabbit secondary antibody was diluted 1∶20,incubating at 37℃for30min.This method had good reproducibility and specificity in B.avium detection.Detection results of chick tissues infected with B.avium showed that this method could specifically localizeB.aviumin the tissues and organs of chicks.Specific fluorescence signals could be found in tracheas lung,liver,heart,spleen and kidney.It had coincidence rate of 99.27% with bacterial isolation and culture.The results showed that the established indirect fluorescent immunohistochemistry could be used as an effective method in research of B.aviumpathogenic mechanism.

关 键 词：间接免疫荧光组化 禽波氏杆菌 定位检测 组织器官 

分 类 号：S852.61[农业科学—基础兽医学]