以合成肽为抗原检测PRRSV抗体ELISA方法的建立及初步应用  

Establishment and Preliminary Application of ELISA Method of PRRSV Antibody by Using Synthetic Peptides as Antigen

作　　者：孙见 SUN Jian(Weihai Ocean Vocational College,Rongcheng 264300,China;Institute of Special Economic Animal and Plant Sciences,CAAS,Changchun 132109,China)

机构地区：[1]威海海洋职业学院,荣成264300 [2]中国农业科学院特产研究所,长春132109

出　　处：《中国动物传染病学报》2022年第4期190-196,共7页Chinese Journal of Animal Infectious Diseases

基　　金：山东省海洋生物健康促进产业职业教育集团重点资助项目(HYSW2101)。

摘　　要：通过软件预测猪繁殖与呼吸综合征病毒(PRRSV)N蛋白的主要抗原性表位,利用化学合成的方法合成三条针对N蛋白抗原表位的多肽作为包被抗原,筛选最优的一条建立了检测PRRSV抗体的间接ELISA方法。该ELISA方法的最适条件为:以2.5μg/mL的合成肽(Pep3)包被酶标板,4℃过夜,不需封闭,阴阳性血清分别稀释200倍,37℃作用60 min,辣根过氧化酶(HRP)标记兔抗猪酶标二抗1000倍稀释,37℃作用30 min,TMB 37℃显色15 min,2 mol/L H_(2)SO_(4)终止反应,酶标仪A(450 nm)读数。利用所建立的方法测定50份阴性血清的值计算S/P值,求得当S/P值≥0.324时,即为阳性,0.324>样品S/P值>0.212即为可疑,S/P值≤0.212时,即为阴性。之后进行了灵敏度试验,将标准阳性血清进行1∶3200倍稀释仍然呈现阳性,说明该检测方法具有较高的灵敏性;与常见的PCV2、CSFV、PRV、SIV 4种猪疫病阳性血清无交叉反应,说明该检测方法具有较高的特异性;通过对临床的200份样品进行检测,与IDEXX-PRRSV抗体检测试剂盒相比较,符合率达91.4%,说明该校测方法具有较高的符合率。本研究建立的ELISA方法对于PRRSV的常规诊断及流行病学调查具有重要意义。The main antigenic epitope of the N protein of swine reproductive and respiratory syndrome virus(PRRSV)protein was predicted by software analysis,three peptides against the N protein epitope were synthesized as coated antigens,and the best indirect ELISA method for detecting PRRSV antibody was selected.The optimal conditions for the ELISA method is 2.5μg/mLsynthetic peptide(Pep3)coated with microplate,4℃overnight,200 times dilution,37℃,horseradish peroxidase(HRP)labeled 1000 times dilution,37℃for 30 min,TMB37℃color 15 min,2 M H_(2)SO_(4) termination reaction,microplate reader A(450 nm)reading.The S/P value was calculated using the value of 50 negative sera,so that when S/P value of 0.324 is positive,0.324>sample S/P value>0.212 is suspicious,and S/P value of 0.212 is negative.the sensitivity test was conducted,and the standard positive serum was diluted 1∶3200 times still positive,which indicates the high sensitivity;no cross-reaction with the positive serum of PCV2,CSFV,PRV and SIV,indicating high specifi city;through 200 clinical samples,compared with the IDEXX PRRSV antibody detection kit,the compliance rate reached 91.4%,It shows that the calibration method has a high compliance rate.The ELISA method established in this study is important for the routine diagnosis and epidemiological investigation of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 N蛋白 抗原表位 合成肽 

分 类 号：S858.28[农业科学—临床兽医学]