猪流行性腹泻病毒N重组表达蛋白间接ELISA检测方法建立  

Development of an Indirect ELISA Method for Detecting Antibody Against Porcine Epidemic Diarrhea Virus Based on Recombinant N Protein

作　　者：顾昀[1,2] 周晓丽[2] 袁秀芳[2] 杜晓莉[2] 徐丽华[2] 李军星[2] 方维焕[1] 王一成[2] 

机构地区：[1]浙江大学,浙江杭州310027 [2]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《华北农学报》2015年第1期123-129,共7页Acta Agriculturae Boreali-Sinica

基　　金：浙江省三农六方项目(2012R22A60C01);浙江省重大科技专项重点农业项目(2012C12009-1)

摘　　要：为了检测猪血清中猪流行性腹泻病毒抗体水平,用纯化的猪流行性腹泻病毒N基因重组表达蛋白作为包被抗原,建立了检测猪流行性腹泻病毒抗体的间接ELISA方法。ELISA的最佳工作条件是:抗原包被浓度为0.5μg/m L,包被时间4℃过夜,5%脱脂乳的PBS封闭37℃2 h及4℃过夜。血清稀释度为1∶100;37℃作用45 min,辣根过氧化物酶标记兔抗猪Ig G稀释度为1∶10 000,37℃温育60 min,底物显色37℃15 min。抗体临界值为OD450nm≥0.30判为阳性,OD450nm≤0.27判为阴性,介于二者之间判为可疑。经重复性试验和交叉试验,结果表明,该方法特异性强、灵敏度高、重复性好。用已建立的ELISA方法检测临床血清样本184份,总阳性率为68.5%。In order to determine the level of antibody against PEDV in swine serum .The PEDV recombinant N protein was expressed by E.coli and purified through native conditions purification method .Based on purified re-combinant N protein ,an indirect ELISA for detection of anti-PEDV antibodies was developed and its optimal reac-tion conditions were determined：antigen working concentration was 0.5 μg/mL,it was coated at 37 ℃for 2 h and 4 ℃overnight,serum samples dilution was 1∶100,incubated at 37 ℃for 45 min.HRP-labeled rabbit anti-pig IgG was diluted at 1∶10 000 ,incubated at 37 ℃for 60 min,the substrate for showing color was incubated at 37 ℃for 15 min.It was judged to be positive when the OD was greater than the cut off OD 450nm of 0.30,as negative when OD450nm was smaller than 0.27,and as suspicious between 0.27 and 0.30.The established ELISA assay was confirmed to be good in specificity,sensitivity and repeatability by repeatability and cross test .A total of 184 clinical serum samples obtained from pig farms in Zhejiang Province were detected by using the established ELISA and the positive rate was 68.5%.

关 键 词：猪流行性腹泻病毒 重组N蛋白 ELISA 

分 类 号：S828[农业科学—畜牧学]