摘要
为建立一种准确检测猪繁殖与呼吸综合征病毒(PRRSV)抗体的方法,通过对PRRSV基因连续缺失片段dNsp2(87)进行诱导表达,重组pUC57-dNsp2蛋白,经镍柱纯化后,作为抗原包被微量反应板,建立了检测PRRSV抗体的间接ELISA方法。经反应条件优化,确定抗原的最适包被浓度为40μg/mL,最适封闭液为5%脱脂奶粉,检测血清稀释度为1:40,酶标二抗的最适工作浓度为1:5 000,最佳显色时间为1 h,确定阴阳性临界值为0.35(OD450)。该方法均不与猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪圆环病毒(PCV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病毒(PRV)阳性血清发生交叉反应,具有良好的特异性。与IDEXX PRRSV Kit进行重复性、敏感性、符合性试验,证明该方法具有较好的重复性、敏感性、符合性。结果表明,该方法可用于临床PRRSV抗体的检测。
In order to develop a method for auurately detecting the antibodies against the porcine reproductive and respiratory syndrome virus (PRRSV) , the dNsp2 (87) gene was amplified, and recombinant pUC57-dNsp2 protein was induced and expressed. Using the purified dNsp2 (87) protein as coating antigens, an indirect ELISA method was established. After optimization of reaction conditions, the optimal concentration of coating antigens was determined to be 40 gg/mL, the optimal blocking solution was 5% skim milk, the serum dilution was 1:40, and the optimal working concentration of the enzyme-labeled secondary antibody was 1:5000, the reaction time of the substrate was 1 h. The cutoff value was 0.35 (OD450) . Using the established method to detect the positive sera against CSFV, PPV, PCV2, JEV and PRV, no cross-action was observed, indicatings good specificity. After comparison with IDEXX PRRSV kit, results showed the developed method had a good reproducibility, high sensitivity and conformity. As a conclusion, the indirect ELISA method could be used in clinical detection of antibodies against PRRSV.
作者
高许雷
孙国强
李永锋
Gao Xulei;Sun Guoqiang;Li Yongfeng(Laoshan District Agriculture and Conservancy Bureau, Qingdao, Shandong 266061, China;Qingdao Agriculture University, Qingdao, Shandong 266109, China;State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang 150069, China)
出处
《中国动物检疫》
CAS
2018年第11期75-78,共4页
China Animal Health Inspection
关键词
高致病性猪繁殖与呼吸综合征病毒
基因连续缺失片段
间接ELISA
highly pathogenic reproductive and respiratory syndrome virus
fragment with continuous gene deletion
indirect ELISA
