摘要
为建立猪流行性腹泻病毒(PEDV)血清学ELISA抗体检测方法,本研究以原核表达纯化的PEDV S2重组蛋白为包被抗原,通过反应条件优化,建立了一种PEDV重组S2蛋白的间接ELISA抗体检测方法。结果显示,重组蛋白抗原最佳包被量0.8μg/孔;血清最佳稀释倍数为1∶40,最佳血清孵育时间为120 min;酶标二抗最佳工作浓度为1∶4000,最佳二抗反应时间为45 min;最佳底物显色时间为15 min;待检血清样品S/P值>0.212时判定为阳性,待检血清样品S/P值<0.188时判定为阴性,当0.188≤待检血清样品S/P值≤0.212时判定为可疑。以S2蛋白作为包被抗原建立的PEDV抗体间接ELISA方法仅对PEDV血清检测为阳性,与猪瘟病毒、猪丁型冠状病毒等主要猪源病毒阳性血清均无交叉反应,具有良好的特异性。该方法检测灵敏度较高、重复性好,批内和批间重复性变异系数均小于10%。利用本研究建立的PEDV S2蛋白间接ELISA抗体检测方法对广西地区不同阶段猪群血清样品共计622份进行检测,总体样品阳性率为76.05%,不同阶段阳性率相差较大。本实验建立的ELISA方法可应用于临床样品PEDV抗体检测以及PEDV血清流行病学调查。
In the present study,the purified PEDV S2 recombinant protein obtained from prokaryotic express system was used as the coating antigen for developing an indirect ELISA antibody detection method.The reaction parameters were optimized as follows:coating antigen of 0.8 μg /well,1:40 dilution of serum and incubation for 120 min,1:4000 dilution of HRP-conjugate and incubation for 40 min,and final incubation of substrate TMB for 15 min under room temperature.The serum samples with S/P >0.212 were determined to be negative,S/P <0.188 to be positive and S/P between 0.188 and 0.212 to be suspicious.The ELISA method was specific for PEDV as it had no cross-reaction with other serum samples against PDCoV,PRRSV,CSFV,PCV-II,FMDV and PRV.The ELISA was highly reproducible with the coefficients of variation at less than 10%.Total 622 clinical serum samples collected from pigs at different ages in Guangxi province were tested for the presence of PEDV antibodies by this indirect ELISA and the positive rate was 76.05% with some ages difference.In brief,the ELISA method developed in the present study could be used for detection of PEDV antibodies for the serologically investigation.
作者
秦毅斌
刘磊
卢冰霞
段群棚
何颖
陈忠伟
周英宁
李斌
赵硕
梁家幸
苏乾莲
蒋冬福
赵武
QIN Yibin;LIU Lei;LU Bingxia;DUAN Qunpeng;HE Ying;CHEN Zhongwei;ZHOU Yingning;LI Bin;ZHAO Shuo;LIANG Jiaxing;SU Qianlian;JIANG Dongfu;ZHAO Wu(Guangxi Key Laboratory of Veterinary Biotechnology/Guangxi Veterinary Research Institute,Nanning 530001,China;Guangxi Agricultural Vocational and Technical College,Nanning 530007,China;Guangxi University,Nanning 530005,China)
出处
《中国动物传染病学报》
CAS
北大核心
2022年第5期74-82,共9页
Chinese Journal of Animal Infectious Diseases
基金
广西创新驱动发展专项资金项目(桂科AA17204057)
广西自然科学基金项目(2017GXNSFBA198092)
广西基本科研业务费专项(桂科专项17-2,19-2)
广西水产畜牧科技项目(201633041,201633034)
广西兽医生物技术重点实验室开放基金课题(16-380-45-B-3,19-50-40-B-01)
南宁市西乡塘区科技项目(201810204)。
关键词
猪流行性腹泻病毒
S2基因
ELISA
Porcine epidemic diarrhea virus
S2 gene
ELISA
detection
antibody
