基于G_(NS)蛋白的牛流行热病毒感染与免疫鉴别诊断ELISA方法的建立  

Development of a Differential Diagnostic ELISA Based on G NS Protein to Distinguish BEFV Infected and Vaccinated Cattle

作　　者：何辰香 高闪电[1] 田占成[1] 独军政[1] 王锦明[1] 关贵全[1] 殷宏[1,2] HE Chenxiang;GAO Shandian;TIAN Zhancheng;DU Junzheng;WANG Jinming;GUAN Guiquan;YIN Hong(State Key Laboratory for Animal Disease Control and Prevention,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所,动物疫病防控全国重点实验室,兰州730046 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《畜牧兽医学报》2023年第10期4320-4326,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：甘肃省重点研发计划(22YF7NA030);甘肃省创新引导计划-科技特派团专项(22CX8NA011);兰州市科技计划项目(2021-1-12);国家重点研发计划项目(2021YFD1800500);甘肃省基础研究创新群体项目(22JR5RA024);国家肉牛牦牛产业技术体系(NBCIS,CARS-37);农业科技创新工程(CAAS-ASTIP-2016-LVRI)。

摘　　要：旨在建立牛流行热病毒(BEFV)感染与疫苗免疫的抗体ELISA诊断方法。在证实BEFV G_(NS)截短体与BEFV感染血清特异性反应的基础上,利用原核表达系统表达纯化G_(NS)全长蛋白并以其作为包被抗原,优化反应条件,建立基于BEFV G_(NS)的鉴别病毒感染与疫苗免疫的BEFV间接ELISA方法。结果显示:在大肠杆菌中成功表达G_(NS)蛋白,主要以包涵体的形式存在,纯化获得73 ku的重组蛋白。Western blot结果显示,纯化后的重组蛋白与BEFV感染牛血清反应原性良好,且与BEF疫苗免疫牛血清未见反应。G_(NS)抗原最佳包被浓度为0.50μg·mL^(-1),血清最佳稀释度为1∶20,酶标二抗最佳稀释度为1∶4000,阴阳性临界值为S/P值0.2069。该方法与BVDV、FMDV以及IBRV阳性血清无交叉反应,特异性良好;批内和批间变异系数均小于10%,重复性良好,可用于BEFV感染与疫苗免疫牛的鉴别诊断。This study focused on developing an antibody-detecting ELISA capable of distinguishing BEFV infected and vaccinated animals.Based on the identified specific reactivity between the truncated BEFV G NS protein and sera from BEFV infected cattle,the full-length G NS recombinant protein was expressed in prokaryotic system,purified and used for developing an antibody-detecting ELISA to distinguish BEFV infected and vaccinated animals under the optimized reaction conditions.The G NS was expressed in Escherichia coli and deposited mainly in inclusion bodies.The purified recombinant protein had a molecular weight of 73 ku.Western blot demonstrated that sera from BEFV infected cattle but not sera from BEF vaccine immunized cattle reacted strongly with the purified G_(NS)protein.The optimal concentrations of plate-coating antigens and the dilution of sera were 0.50μg·mL^(-1)and 1∶20,the HRP conjugated secondary antibody was determined to be 1∶4000,and the S/P ratio=0.2069 was selected as the negative/positive cut-off value.Acceptable specificity and repeatability were confirmed by no cross reactivity with positive sera of BVDV,FMDV and IBRV as well as coefficient of variation less than 10%in intra-batch and inter-bath.The DIVA(differentiation of infected from vaccinated animals)ELISA was developed and can be used for distinguish BEFV infected and vaccinated cattle.

关 键 词：牛流行热 牛流行热病毒 牛流行热灭活疫苗 G_(NS)蛋白 鉴别诊断ELISA 

分 类 号：S852.659.5[农业科学—基础兽医学]