猪伪狂犬病病毒IgA抗体间接ELISA检测方法的建立  

Development of an indirect ELISA for the detection of IgA antibodies against porcine pseudorabies virus

作　　者：黄雨晴 华涛[2,3,4,5] 唐波 常晨[2,3,4,5] 刘国阳 张雪花[2,3,4,5] 卢宇 侯继波[2,3,4,5] 张道华 许家荣[1] HUANG Yu-qing;HUA Tao;TANG Bo;CHANG Chen;LIU Guo-yang;ZHANG Xue-hua;LU Yu;HOU Ji-bo;ZHANG Dao-hua;XU Jia-rong(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;National Research Center of Engineering and Technology for Veterinary Biologicals,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Key Lab of Food Quality and Safety of Jiangsu Province—State Key Laboratory Breeding Base,Nanjing 210014,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [3]江苏省农业科学院国家兽用生物制品工程技术研究中心,江苏南京210014 [4]省部共建国家重点实验室培育基地—江苏省食品质量安全重点实验室,江苏南京210014 [5]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2019年第11期1346-1353,共8页Chinese Veterinary Science

基　　金：江苏省农业自主创新项目[cx（17）3049]

摘　　要：为了建立检测猪伪狂犬病病毒(PRV)黏膜免疫时特异的IgA抗体的间接ELISA方法,本研究采用PCR方法扩增了PRV gB基因截短片段,并将其连入原核表达质粒pGEX-6P-1,构建的表达质粒转化至大肠杆菌Transetta(DE3)菌株后,采用IPTG进行诱导表达,得到gB重组蛋白的可溶性表达,并用谷胱甘肽亲和层析柱进行蛋白纯化。以纯化的gB重组蛋白作为包被抗原,以山羊抗猪IgA-HRP为二抗,建立了猪伪狂犬病病毒IgA的间接ELISA检测方法。经ELISA反应条件优化,确定抗原包被浓度为2.4μg/mL,封闭液为10 g/L明胶,一抗37℃孵育1.5 h,二抗1∶20 000稀释后37℃孵育0.5 h,TMB显色液显色10 min。该方法的批内和批间变异系数均小于10%,重复性较好。利用该间接ELISA方法对PRV黏膜免疫的仔猪进行检测,结果可以在鼻腔黏液中检测出特异的IgA抗体。本试验为PRV黏膜免疫提供了初步检测方法。To establish a method for the detection of IgA antibodies against porcine pseudorabies virus(PRV),an indirect ELISA(i ELISA)was developed.The truncated gB gene fragment of PRV was designed and amplified by PCR and cloned into the prokaryotic expression plasmid p GEX-6 P-1.The recombinant plasmid was transformed into E.coli Transetta(DE3) strain and induced by IPTG,and then the recombinant gB protein was obtained in a soluble form.Furthermore,the recombinant gB protein was purified by glutathione affinity chromatography column.The purified recombinant gB protein was used as the coating antigen,and the HRP-conjugated goat anti-pig IgA was used as the secondary antibody.The optimized reaction conditions showed that the optimal antigen coating concentration was 2.4μg/m L,the optimal blocking liquid was 10 g/L gelatin,the primary antibody incubate for 1.5 h at 37 ℃,the optimal working dilution of secondary antibody was 1 ∶ 20 000,the secondary antibody incubate for 0.5 h at 37 ℃,and the optimal working time of chromogen solution was 10 min.The coefficient of variation of reproducibility was less than 10%.The i ELISA was used to detect IgA antibodies against porcine pseudorabies virus from piglets immunized with PRV by mucosal immune pathway,Ig A antibodies can be detected in nasal mucus.In conculsion,this method provides a preliminary detection method for PRV mucosal immunity.

关 键 词：伪狂犬病病毒 GB基因 原核表达 IgA 间接ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]