牛病毒性腹泻病毒E_(60~145aa)^(rns)片段可溶表达及抗体间接ELISA检测方法的建立  

Soluble Expression of Bovine Viral Diarrhea Virus E_(60~145aa)^(rns) Fragment and Establishment of Indirect ELISA Method for Antibody Detection

作　　者：张宇名 李树凡 陈志远 迟丽丽 刘健 于泽海 张玫瑜 康京丽[2] 徐守振[1] Zhang Yuming;Li Shufan;Chen Zhiyuan;Chi Lili;Liu Jian;Yu Zehai;Zhang Meiyu;Kang Jingli;Xu Shouzhen(College of Animal Science and Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;China Center for Animal Health and Epidemiology,Qingdao 266032,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109 [2]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《山东农业科学》2022年第11期127-137,共11页Shandong Agricultural Sciences

基　　金：国家重点研发计划项目(2018YFD0501403)。

摘　　要：为建立牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体间接ELISA检测方法,本研究对山东地区规模化奶牛场BVDV主要流行亚型的E^(rns)蛋白进行表位分析,原核可溶表达优势抗原表位片段E_(60~145aa)^(rns),经Western blot及LC-MS/MS鉴定后作为ELISA包被抗原,对影响ELISA试验的各个因素进行优化,并进行特异性、敏感性、重复性试验等。结果显示,间接ELISA方法优化后的条件为:0.25μg/mL抗原包被37℃2 h后4℃过夜,10%马血清封闭时间1.5 h,1∶5稀释一抗孵育1.5 h,1∶8000稀释二抗孵育60 min,显色25 min。该方法特异性、灵敏性及重复性良好,与商品化BVDV抗体检测试剂盒总体符合率为88.04%,符合率良好。本研究建立了一种特异性、灵敏性及重复性良好的BVDV抗体间接ELISA检测方法,为山东地区BVDV防控及临床检测提供了一种有效工具。To establish an indirect ELISA detection method for bovine viral diarrhea virus(BVDV)antibody,the epitope analysis of BVDV E^(rns) protein from dominant epidemic subtype was conducted,and the prokaryotic soluble expression of dominant epitope fragments E_(60~145aa)^(rns)was as ELISA coated antigens after identified by Western blot and LC-MS/MS.The factors influencing the ELISA test were optimized,and the tests on specificity,sensitivity and repeatability were carried out.The results showed that the optimal conditions for indirect ELISA were as follows:0.25μg/mL antigen coated at 37℃for 2 h and overnight at 4℃,10%equine serum sealed for 1.5 h,primary antibody diluted at 1∶5 and incubated for 2.5 h,second antibody diluted at 1∶8000 and incubated for 60 min,and colored for 25 min.The established indirect ELISA method had good specificity,sensitivity and repeatability.Its overall coincidence rate with the commercial BVDV antibody detection kit was better as 88.04%.In conclusion,an indirect ELISA method with better specificity,sensitivity and reproducibility was established for detecting BVDV antibody in this study,which could be an effective tool for BVDV prevention and control and clinical detection in Shandong Province.

关 键 词：牛病毒性腹泻病毒 Erns蛋白 抗体 间接ELISA 

分 类 号：S852.653[农业科学—基础兽医学]