以原核表达的猪δ冠状病毒N蛋白为包被抗原的间接ELISA方法的建立  

Establishment of a recombinant nucleoprotein-based ELISA for detection of antibodies against newly emerged porcine deltacoronavirus

作　　者：张帆帆[1] 宋德平[1] 郭楠楠[1] 叶昱[1] 周信荣[1] 李安琪[1] 张敏[1] 彭棋 陈燕君[1] 黄冬艳[1] 唐玉新[1] 

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045

出　　处：《中国预防兽医学报》2016年第10期795-799,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31372457);江西省科技厅落地计划项目(KJLD13029)

摘　　要：为建立检测血清中猪δ冠状病毒(PDCoV)抗体的间接ELISA方法,本研究根据PDCoV的N基因序列设计特异性引物,扩增出N蛋白全基因序列,将其克隆至低温表达载体p Cold I中并转入BL21,诱导表达获得大小约为41 ku的可溶性目的蛋白;western blot试验表明所表达的蛋白具有反应活性。以纯化的重组N蛋白作为包被抗原,建立了PDCoV间接ELISA抗体检测方法。结果表明,本研究建立的ELISA方法阴阳性临界值为0.316,与猪流行性腹泻病毒等6种猪常见病原阳性血清均无交叉反应,具有良好的特异性。批内和批间重复性试验的变异系数均小于10%,重复性和稳定性均较好。应用建立的间接ELISA方法,对收集自江西各地区282份猪血清进行检测,阳性率为12.8%(36/282),表明PDCoV在我省猪群中普遍存在。本实验建立的PDCoV抗体捕获间接ELISA为临床上猪群PDCoV感染监测和流行病学调查提供了实验依据。To establish an assay for detection of the serum antibodies against porcine deltacoronavirus （PDCoV）, the complete nucleotide （N） protein gene of newly emerged PDCoV was amplified by a RT-PCR with a pair of specific primers targeting the N gene of PDCoV and cloned into a low temperature-induced expression vector pCold I, and then the soluble N proteins of PDCoV with the size about 41 ku were expressed in E.coli. The reactionogenicity of the expressed protein was tested and assessed by western blot. An expressed N protein-based ELISA was then established and evaluated. The results showed that the established ELISA was highly sensitive and specific, repetitive and stabile. A total of 282 swine sera were tested for the presence of antibodies against PDCoV. Thirty six out of 282 （12.8%） sera tested were positive for PDCoV antibody, indicating that the PDCoV was wide prevalence in among pigs in Jiangxi. This study provides a useful tool for routine clinic diagnostics, epidemiological surveillance, and outbreak investigations.

关 键 词：猪δ冠状病毒 N蛋白 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]