基于重组截短RHDV VP60蛋白的间接ELISA抗体检测方法  

Indirect ELISA method based on recombinant truncated VP60 protein for the detection of RHDV antibodies

作　　者：孙培姣 周迎春[3] 于雪 李本科 王辉暖 王雅雯 于新友 杨慧 王蓉[5] 李振伟 王玉茂[1] 沈志强[1,2] 肖跃强 SUN Peijiao;ZHOU Yingchun;YU Xue;LI Benke;WANG Huinuan;WANG Yawen;YU Xinyou;YANG Hui;WANG Rong;LI Zhenwei;WANG Yumao;SHEN Zhiqiang;XIAO Yue-qiang(Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou,Shandong 256600,China;Shandong Lyudu Bio-science&Technology Co.Ltd,Binzhou,Shandong 256600,China;Anhui Provincial Center for Animal Diseases Control and Prevention,Hefei 230061,China;Binzhou Animal Husbandry and Veterinary Management Service Center,Binzhou 3Shandong 256600,China;School of Basic Medical Sciences,XVan Jiaotong University,Xi'an 710061,China;College of Animal Husbandry&Veterinary Medicine,Jinzhou Medical University,Jinzhou,Liaoning 121000,China)

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600 [3]安徽省动物疫病预防与控制中心,安徽合肥230061 [4]滨州市畜牧兽医管理服务中心,山东滨州256600 [5]西安交通大学基础医学院,陕西西安710061 [6]锦州医科大学畜牧兽医学院,辽宁锦州121000

出　　处：《中国兽医学报》2021年第7期1264-1269,共6页Chinese Journal of Veterinary Science

基　　金：山东省自然科学基金资助项目(ZR2014CQ045);山东省现代农业产业技术体系资助项目(SDAIT-21-15);陕西省自然科学基础研究计划(2019JQ-599);滨州市科技发展计划资助项目(2015ZC0108)。

摘　　要：为了建立一种快速、特异、敏感的RHDV抗体检测方法,对VP60蛋白基因的抗原表位区域进行分析,截取VP60-1、VP60-2、VP60-3、VP60-semi1和VP60-semi2,大肠杆菌表达后纯化获得重组截短蛋白。经抗原、抗体结合反应比较,以VP60-semi1为包被抗原检测效果最好,并建立了基于重组蛋白VP60-semi1的间接ELISA抗体检测方法。经优化,该方法的最佳条件:抗原包被质量浓度为0.1μg/孔,4℃包被过夜;5%脱脂奶粉37℃封闭1 h;血清稀释度为1∶200,37℃孵育1 h;最后37℃作用显色15 min。该方法与血凝抑制(HI)检测相比,敏感性好,阳性血清稀释至1∶6 400仍可检出;批内、批间变异系数分别为0.46%~4.97%和2.68%~8.88%,重复性好;符合率检测显示,与HI法的符合率为89.29%,与免疫保护试验结果符合率为100%。结果表明,建立了一种基于重组蛋白VP60-semi1的间接ELISA抗体检测方法,可用于监测兔群免疫后抗体水平动态并指导疫苗免疫,以及该病的流行病学调查等。In order to establish a rapid, specific and sensitive method for detecting rabbit hemorrhagic disease virus(RHDV) antibodies, the regions of VP60 protein containing the antigenic epitopes were analyzed, through which the VP60 was truncated into five fragments designated as VP60-1,VP60-2,VP60-3,VP60-semi1 and VP60-semi2,and the proteins were expressed in E.coli.Through antigen-antibody binding test, the recombinant protein VP60-semi1 was selected for the indirect ELISA antibody detection, and an indirect ELISA method based on VP60-semi1 was established.The optimized conditions of this method as follows: the protein coating amount was 0.1 μg/well and plates were coated at 4℃ overnight, the regent for sealing was 5% skimmed milk powder solution and plates were sealed at 37℃ for 1 h,the serum dilution was 1∶200 and incubated at37℃for 1 h,catalytic reaction of TMB substrate by HRP was carried out at 37℃for 15 min.The sensitivity of this method was higher than the hemagglutination inhibition(HI)test,and the detectable dilution of the positive serum reached to 1∶6 400.Repeatability and reproducibility test of this method were very high,the intra-and inter-assay coefficient of variation were 0.46%-4.97%and 2.68%-8.88%,respectively.The coincidence rate test showed that the compliance rates with the HI test and immune protection test were 89.29% and 100%,respectively.From the results above mentioned,this method can be used to assess the immune status of rabbits,monitor the antibody duration after vaccination,conduct vaccine administration and fulfill epidemiological investigation.

关 键 词：RHDV 截短VP60蛋白 间接ELISA方法 抗体检测 

分 类 号：S852.65[农业科学—基础兽医学]