基于H6蛋白禽流感抗体间接ELISA检测方法建立与应用  

Establishment and Application of Indirect ELISA Detection Method for Avian Influenza Type H6 Antibody Based on H6 Protein

作　　者：王微 张霞[2] 张人俊[2] 胡安东 杨颖[1,4] 温贵兰 文明[1,4] Wang Wei;Zhang Xia;Zhang Renjun;Hu Andong;Yang Ying;Wen Guilan;Wen Ming(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025,China;Guizhou Center of Animal Disease Prevention and Control,Guiyang,Guizhou 550008,China;Guizhou Vocational College of Agriculture,Qingzhen,Guizhou 551400,China;Guizhou Research Center of Engineering Technology for Animal Biological Products,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病预防控制中心,贵州贵阳550008 [3]贵州农业职业学院,贵州清镇551400 [4]贵州省动物生物制品工程技术研究中心,贵州贵阳550025

出　　处：《山地农业生物学报》2022年第1期21-27,共7页Journal of Mountain Agriculture and Biology

基　　金：贵州省科技支撑计划项目(黔科合支撑[2020]1Y043);贵州省科技支撑计划项目(黔科合支撑[2019]2286);贵州省科技平台建设项目(黔科合平台人才[2018]5253)。

摘　　要：为临床H6亚型禽流感抗体检测提供技术支撑,通过克隆得到的H6亚型禽流感病毒H6基因并与原核表达载体pET-32α连接,经PCR、酶切和测序鉴定后,转化大肠杆菌进行诱导表达和Western blotting鉴定,以纯化的表达产物为包被抗原,通过反应条件优化,建立检测禽流感病毒H6抗体的间接ELISA方法并对临床鸡血清样本检测评价。结果表明:该方法最佳反应条件是,包被抗原浓度为10μg/mL,4℃过夜;5%脱脂奶粉37℃封闭2 h;血清稀释度为1∶20,37℃孵育30 min;酶标二抗稀释度为1∶5000,37℃作用30 min;显色时间为37℃10 min。该ELISA方法可特异性检测H6亚型禽流感抗体,阳性血清稀释至1∶320仍可检出,与其他禽病阳性血清均无交叉反应,检测变异系数均小于10%。以该ELISA方法检测2017年至2019年收集的1837份家禽临床血清样本,H6亚型禽流感抗体阳性率为10.45%。这些结果表明,建立的基于H6蛋白禽流感抗体间接ELISA检测方法,具有良好的特异性、敏感性、重复性和可靠性,能为H6亚型禽流感血清流行病学调查提供技术手段。To provide technical support for clinical avian influenza subtype H6 antibody detection,the H6 gene of avian influenza virus was cloned and connected the prokaryotic expression vector pET-32α.After identification by PCR,double enzyme digestion and sequencing,the positive recombinant plasmid was transformed into E.coli(DE3)for induction expression by IPTG and western blotting identification,with the purified expression product as the coating antigen,through optimization of reaction conditions,an indirect ELISA method for detecting avian influenza virus subtype H6 antibody was established and the clinical chicken serum samples were tested and evaluated.The results were showed as follows:the optimum reaction conditions of indirect ELISA,were 10μg/mL of coated antigen concentration and overnight at 4℃,5%skimmed milk powder sealed at 37℃for 2 h,1∶20 of the serum dilution and 30 min of incubation at 37℃,1∶5000 of HRP-IgG dilution and 30 min of the reaction time at 37℃,10 min of the TMB reaction time at 37℃.The ELISA method could specifically detect the AI H6 antibody but no cross reaction with other avian pathogen positive serum,and the positive serum was still positive after 1∶320 dilution.The variation coefficient of detection was less than 10%.The antibody positive rate was 10.45%against avian influenza subtype H6,which 1837 of serum samples were collected in poultry from 2017 to 2019.These results indicated that the indirect ELISA method based on H6 protein has good specificity,sensitivity,repeatability and reliability,which could provide technical means for serological investigation of avian influenza subtype H6.

关 键 词：H6亚型禽流感 H6蛋白 间接ELISA方法 建立 应用 

分 类 号：S852.65[农业科学—基础兽医学]