猪塞尼卡病毒VP2 ELISA抗体检测方法的建立及初步应用

Development of an indirect ELISA assay for detecting the antibodies against VP2 of Senecavirus A

猪塞尼卡病毒(SVA)是影响我国生猪产业的一种新的重要病原,临床上主要引起猪口腔和蹄冠部水泡,与口蹄疫临床症状十分相似。为建立快速、敏感和特异的SVA血清抗体检测方法,本研究采用原核表达系统表达该病毒VP2蛋白,证实其以包涵体形式表达,纯化后获得了VP2重组蛋白。经过反应条件优化,建立了SVA VP2间接ELISA抗体的检测方法。该方法与口蹄疫病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒和猪伪狂犬病毒阳性血清均无交叉反应;敏感性为91.6%,批内和批间重复系数分别为11.1%和9.67%。上述结果表明该方法特异性、敏感性和重复性较好。符合率试验结果显示,该方法与血清中和抗体试验符合率为91.9%。利用本实验建立的间接ELISA抗体方法检测2016年~2019年华东地区5个省份2070份猪血清,结果显示,江苏、安徽、浙江、山东和江西省SVA抗体阳性率分别为73%、67%、69%、74%和73%;且各年度抗体阳性率均较高,其中2016年最高达86.9%。本研究为我国SVA检测和流行病学调查提供了有效方法。

Senecavirus A(SVA)is a new important pathogen that affects the pigs industry in China,which causes the similar clinical symptoms of foot-and-mouth disease,i.e.,oral and hoof crown blisters in the infected pigs.To develop a sensitive and specific method for detection of the serum antibody against the virus,in this study,the VP2 protein was expressed using the the prokaryotic expression system and was confirmed as inclusion bodies form.The purified VP2 protein was prepared as a coating antigen,and an indirect ELISA was developed and optimized for the detection of anti-VP2 antibodies.This method has no crossreaction with those positive sera samples of foot-and-mouth disease,porcine reproductive and respiratory syndrome,porcine circovirus 2,classical swine fever and porcine rabies viruses.The sensitivity of this method was 91.6%,and the intra-and interassay repeatability coefficients were 11.1%and 9.67%,respectively.The coincidence rate of this method and the SVA serum neutralizing antibody test was 91.9%.2070 swine sera samples collected from the mid-east since 2016 to 2019 were detected using the developed method,and the positive rate in Jiangsu,Anhui,Zhejiang,Shandong,and Jiangxi provinces was 73%,67%,69%,74%, 73%, respectively. The antibody positive rate was high in the swine herd and the highest antibody positive rate was 86.9% in2016. This provides an effective method for the diagnosis and epidemiological investigation of SVA in China.