A型塞内卡病毒3AB蛋白原核表达及间接ELISA方法的初步建立  

Prokaryotic expression of Senecavirus A 3AB protein and preliminary establishment of indirect ELISA detection method

作　　者：郑君佐 张中旺 马中元 梁志博 白英杰 赵永聪 潘丽[2] 包世俊[1] 吕建亮[2] ZHENG Junzuo;ZHANG Zhongwang;MA Zhongyuan;LIANG Zhibo;BAI Yingjie;ZHAO Yongcong;PAN Li;BAO Shijun;LU Jianliang(College of Veterinary Medicine,Gansu A gricultural University,Lanzhou 730070,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院、兰州兽医研究所,甘肃兰州730046

出　　处：《中国兽医科学》2025年第3期338-345,共8页Chinese Veterinary Science

基　　金：财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-35)。

摘　　要：为建立一种检测A型塞内卡病毒(SVA)非结构蛋白(NSP)3AB抗体的间接ELISA方法,本研究利用原核表达系统成功表达了重组SVA3AB蛋白,使用重组SVA3AB蛋白作为包被抗原,建立了间接ELISA方法。结果显示,重组SVA 3AB蛋白以可溶性形式表达,大小为18 k Da,能够与SVA抗体发生特异性反应。建立的间接ELISA方法:抗原最适包被质量浓度为1μg/m L;最佳封闭液为3%脱脂奶粉溶液;待检血清最佳稀释度为1∶640;酶标抗体最佳稀释度为1∶10 000;避光显色时间为10 min。该ELISA方法与猪口蹄疫病毒(FMDV)阳性血清、猪伪狂犬病病毒(PRV)阳性血清、猪繁殖与呼吸综合征病毒(PRRSV)阳性血清、猪瘟病毒(CSFV)阳性血清以及猪圆环病毒(PCV)阳性血清均不发生交叉反应;敏感性达到1∶6 400;批内变异系数小于8%,批间变异系数小于10%。研究结果表明,该间接ELISA方法特异性强,敏感性高,重复性好,可用于临床上猪血清样品的检测,为SVA的鉴别诊断奠定了基础。This study was aimed to establish an indirect ELISA method for detecting the antibodies against non-structural protein(NSP)3AB of Seneca virus A(SVA),the recombinant protein of SVA 3AB was expressed by using a prokaryotic expression system.Using the recombinant protein of SVA 3AB as the coating antigen,an indirect ELISA detection method was established.The results showed that the re-combinant protein of SVA 3AB was expressed in a soluble form with a size of 18 kDa and could specifical-ly react with SVA antibodies.The results of established indirect ELISA method showed that:the optimal coating concentration of antigen was 1µg/mL;the best blocking solution was 3%skimmed milk powder;the optimal dilution ratio of the serum sample was 1:640;the optimal dilution ratio of the enzyme-la-beled antibody was 1∶10000;and in the darkness development time was 10 minutes.This ELISA method did not cross-react with positive sera of porcine foot-and-mouth disease virus(FMDV),porcine pseudora-bies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),and porcine circovirus(PCV);the sensitivity reached 1∶6400;the intra-batch coefficient of variation was less than 8%,and the inter-batch coefficient of variation was less than 10%.This in-direct ELISA method has strong specificity,high sensitivity,and good repeatability.It can be applied or clinical detection of pig serum samples and lays a foundation for the diagnosis of SVA.

关 键 词：猪A型塞内卡病毒 非结构蛋白3AB 原核表达 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]