A型塞内卡病毒VP2蛋白抗原区的原核表达及间接ELISA抗体检测方法的建立  

Prokaryotic expression of VP2 antigen region of type A Seneca virus and establishment of an indirect ELISA antibody detection method

作　　者：翟刚 顾文源[1,3] 王晶[1] 刘莹[1,2] 赵云环 刘涛 张帅[1] 左玉柱[1,2] 范京惠[1,2] ZHAI Gang;GU Wen-yuan;WANG Jing;LIU Ying;ZHAO Yun-huan;LIU Tao;ZHANG Shuai;ZUO Yu-zhu;FAN Jing-hui(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Hebei Agricultural University,Baoding 071001,China;Hebei Center for Animal Disease Prevention and Control,Shijiazhuang 050053,China;Ringpu(Baoding)Biological Pharmaceutical Co.,Ltd.,Baoding 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001 [3]河北省动物疫病预防控制中心,河北石家庄050053 [4]瑞普(保定)生物药业有限公司,河北保定071001

出　　处：《中国预防兽医学报》2023年第10期1025-1031,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省省级科技计划资助(21326611D、19226622D);河北省农业产业技术体系生猪创新团队(HBCT2023170201、HBCT2023170401)。

摘　　要：为建立高效的A型塞内卡病毒(SVA)的血清学检测方法,本研究经PCR扩增SVA VP2基因的优势抗原区域(VP2-1基因)后构建重组质粒p ET-32a-VP2-1,将其转化大肠杆菌感受态细胞并经IPTG诱导表达重组VP2蛋白(rVP2),表达产物经镍柱亲和层析法纯化后利用western blot鉴定,结果显示,在42 ku处出现特异性条带,表明r VP2可以与SVA阳性血清特异性反应。以r VP2为包被抗原,经反应条件的优化,建立了基于SVA VP2抗原区的间接ELISA抗体检测方法。本研究建立的间接ELISA检测方法的最适抗原包被浓度为2μg/mL,5%脱脂乳的PBST作为封闭液37℃孵育1 h,待检血清的最适稀释度为1∶500,兔抗猪Ig G-HRP稀释度为1∶10000,最佳显色时间为10 min。当检测样品的S/P值<0.201时,样品检测结果为阴性;当检测样品的S/P值≥0.201时,结果为阳性。利用该方法检测SVA,猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合症病毒(PRRSV)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)等阳性血清,结果显示,除SVA检测为阳性外,其他病毒均为阴性,特异性较强。将SVA阳性血清2倍倍比稀释(1∶50,1∶100,1∶200,1∶400,1∶800,1∶1600)后经该间接ELISA方法检测,评估其敏感性,结果显示,SVA阳性小鼠血清在1∶800稀释后检测结果仍为阳性,敏感性较高。将同一批次和不同批次纯化的r VP2包被ELISA板,按照该ELISA方法检测8份猪SVA阳性血清,评估该方法的重复性。结果显示,批内、批间重复性试验的变异系数均小于10%,该方法重复性好。利用本实验建立的间接ELISA方法与血清中和试验同时对266份临床血清样品检测,结果显示,本研究建立方法检测的样品阳性率为19.1%,中和试验检测的样品阳性率为19.55%,两种方法检测结果符合率为97.7%。本研究首次基于SVA r VP2建立的间接ELISA方法操作简单、特异性强、敏感性高、重复性好,为SVA的检测和其感染的防控提供了可靠的技术支持。To establish an efficient serological detection method for Seneca virus type A(SVA),in this study,a recombinant plasmid pET-32a-VP2-1 was constructed after PCR amplification of the SVA VP2-1 gene,and the recombinant plasmid was transformed into E.coli competent cells to induce the expression of recombinant VP2 protein(rVP2).The expression product was purified by nickel column affinity chromatography and then identified by western blot.The results showed that the recombinant rVP2 protein could react specifically with SVA-positive serum.An indirect ELISA method based on the SVA VP2 antigen region was established by optimizing the reaction conditions using recombinant rVP2 protein as the encapsulated antigen.The optimum antigen encapsulating concentration of the indirect ELISA method established in this study was 2μg/mL,5%skimmed milk in PBST was used as the blocking solution and incubated at 37℃for 1 hours,the optimum dilution of the serum to be tested was 1:500,and the dilution of rabbit anti-pig IgG-HRP was 1∶10000.The optimum color development time was 10 minutes.When the S/P value of the test sample was<0.201,the sample was defined as negative;when the S/P of the test sample was≥0.201,the sample was defined as positive.The method was used to detect positive sera for SVA,porcine pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine epidemic diarrhea virus(PEDV),and swine fever virus(CSFV).The results showed that,except for the SVA,all the others were negative,with strong specificity.The sensitivity of this method was then evaluated by dilution of SVA positive sera(1∶50,1∶100,1∶200,1∶400,1∶800,1∶1600),and the results showed that the serum of SVA-poistive mouse was still positive when diluted at 1∶800,with high sensitivity.The results of intra-batch and inter-batch repeat tests showed that the coefficient of variation was less than 10%and the method was repeatable.The indirect ELISA method established in this study and serum neutralization test were used to

关 键 词：猪 A型塞内卡病毒 VP2 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]