基于HN蛋白的牛副流感病毒3型间接ELISA检测方法的建立及初步应用  

Establishment and preliminary application of indirect ELISA method for detection of bovine parainfluenza virus type 3 based on HN protein

作　　者：李红 安瑞 李驰欢 朱思萍 董玉来 吴同垒[1] 史秋梅[1] 张志强 LI Hong;AN Rui;LI Chihuan;ZHU Siping;DONG Yulai;WU Tonglei;SHI Qiumei;ZHANG Zhiqiang(Hebei Provincial Key Laboratory of Preventive Veterinary Medicine,College of Animal Science and Technology,Hebei Normal University of Science and Technology,Qinghuangdao,Hebei 066004,China;Xinrui Agricultural Development Co.,Ltd.of Weichang Manchu Mongolian Autonomous County,Chengde,Hebei 068450,China)

机构地区：[1]河北科技师范学院动物科技学院,河北省预防兽医重点实验室,河北秦皇岛066004 [2]围场满族蒙古族自治县新瑞农业开发有限公司,河北承德068450

出　　处：《中国兽医学报》2025年第3期397-403,共7页Chinese Journal of Veterinary Science

基　　金：中央引导地方科技发展资金资助项目(236Z6604G);河北省现代农业产业体系肉牛产业创新团队疫病防控与减抗岗位资助项目(HBCT2024240201);河北省高层次人才资助项目(C20231014);河北科技师范学院高校基本科研任务业务费专项资助项目(2023JK14);承德国家可持续发展议程创新示范区建设科技专项资助项目(202302F040);河北省自然科学基金资助项目(C2024407005)。

摘　　要：为建立牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)血清学检测方法,本试验对BPIV3的HN、NP、F、P蛋白进行原核表达、纯化,筛选最适蛋白包被抗原,建立间接ELISA检测方法。结果显示,成功表达了BPIV3的4种重组蛋白rHN、rNP、rF和rP;棋盘滴定结果显示,rHN蛋白作为包被蛋白具有最高的P/N值,因此用于后续方法建立。摸索间接ELISA的最适反应条件:抗原包被质量浓度为0.5 mg/L,37℃1.5 h;5%脱脂牛奶,4℃过夜封闭;血清1∶50稀释,37℃孵育1 h;二抗稀释度为1∶10000,37℃孵育0.5 h;底物反应条件为37℃12 min。特异性试验结果显示,所建立的方法能够特异性识别BPIV3抗体阳性血清,且灵敏度达到1∶800,批内与批间重复性的检测中变异系数均小于10%,与SVANOVIR试剂盒检测同一批样品的总符合率为92.22%。应用该方法对河北地区192份血清样品进行检测,血清中BPIV3抗体阳性率为66.15%。结果表明,本试验构建的BPIV3抗体间接ELISA检测方法适用于临床大规模血清学调查。In order to establish a serological method for the detection of bovine parainfluenza virus type 3(BPIV3),the prokaryotic expression and purification of BPIV3 HN,NP,F,and P proteins were carried out,and the optimal protein-coated antigen was screened,and an indirect ELISA detection method was established.The results showed that the four recombinant proteins of BPIV3,rHN,rNP,rF,and rP were expressed,and the checkerboard titration results showed that rHN protein had the highest P/N value as the coating protein,so it was used for the subsequent method establishment.The optimal reaction conditions for indirect ELISA were found to be:the mass concentration of the antigen coating was 0.5 mg/L,37℃1.5 h,5%skim milk,overnight blocking at4℃,serum dilution at 1:50,incubation at 37℃1 h,secondary antibody dilution at 1:10000 and incubation at 37℃0.5 h,substrate reaction conditions were 37℃for 12 min.The results of specificity experiments showed that the established method could specifically identify BPIV3 antibodypositive serum with a sensitivity of 1:800,and the coefficient of variation in the detection of intraand inter-assay repeatability was less than 10%,and the overall coincidence rate of the same batch of samples detected with the SVANOVIR kit was 92.22%.This method was used to detect 192 serum samples in Hebei Province,and the positive rate of BPIV3 antibody in serum was 66.15%.The indirect ELISA detection method of BPIV3 antibody constructed in this study is suitable for largescale clinical serological investigations,and provides valuable data support for the research and development of BPIV3 antigen and antibody detection kits in China.

关 键 词：牛副流感病毒3型(BPIV3) HN蛋白 间接ELISA 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]