猪圆环病毒2型Rep抗体间接ELISA检测方法的建立  

Establishment of Indirect ELISA Detection Method for Rep Antibody of Porcine Circovirus Type 2

作　　者：陈清清 荣俊[1,2] 徐保娟 Chen Qingqing;Rong Jun;Xu Baojuan(School of Life Sciences,Yangtze University,Jingzhou 434025,Hubei;Institute of Biomedicine,Yangtze University,Jingzhou 434025,Hubei;Qingdao Yibang Bioengineering Co.Ltd.,Qingdao 266114,Shandong)

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]长江大学生物医药研究所,湖北荆州434025 [3]青岛易邦生物工程有限公司,山东青岛266114

出　　处：《长江大学学报（自然科学版）》2020年第4期77-86,I0007,共11页Journal of Yangtze University(Natural Science Edition)

基　　金：高等教育综合奖基金项目“猪圆环病毒2型鉴别诊断试剂盒的研究”(2018-702090250401)。

摘　　要：猪圆环病毒2型(porcine circovirus type 2,PCV2)是一种广泛存在于猪体内的DNA病毒。养猪场中猪的PCV2感染率较高,为区分自然感染猪和疫苗免疫猪,以大肠杆菌表达的PCV2 Rep蛋白为包被抗原,通过优化反应条件建立PCV2 Rep抗体检测的间接ELISA方法。原核表达的Rep蛋白大小40ku,经过变性亲和层析纯化后目的蛋白纯度为95.1%,批次内和批次间血清检测变异系数均低于10%,表明Rep-ELISA具备良好的稳定性。血清学交叉试验显示Rep-ELISA具有良好的特异性;猪免疫试验结果表明Rep-ELISA能检测出PCV2全病毒疫苗免疫以及接种PCV2 SD/2017活毒的猪血清中的Rep抗体,而不能与PCV2亚单位疫苗免疫猪血清中的抗体发生免疫结合反应。研究结果表明,所建立的ELISA方法能有效鉴别自然感染或全病毒灭活疫苗免疫猪与亚单位疫苗免疫猪,可用于猪群中PCV2的净化。Porcine circovirus type 2(PCV2)is a widespread DNA virus in pigs.Due to the high PCV2 infection rate in pig farms,in order to distinguish naturally infected pigs from vaccine-immunized pigs,an indirect ELISA method for the detection of PCV2 Rep antibody was established by optimizing the reaction conditions with PCV2 Rep protein expressed by Escherichia coli as the coating antigen.The results demonstrate that the Rep protein expressed by pronucleus is more than 40ku and the purity of the target protein is 95.1%after purification by denaturing affinity chromatography.The variation coefficients of serum test within and among the batches are all less than 10%,indicating that the Rep-ELISA has good stability.The serological cross test shows that Rep-ELISA has good specificity.The results of pig immunization experiments show that the Rep-ELISA can detect Rep antibody in pig serum immunized with PCV2 whole virus vaccine and vaccinated with PCV2 SD/2017,but cannot produce immune binding reaction with the antibody in pig serum immunized with PCV2 subunit vaccine.The results also show that the established ELISA method can effectively distinguish naturally infected or whole-virus inactivated vaccine immunized pigs from subunit vaccine immunized pigs,and can be used for elimination of PCV2 in pig herds.

关 键 词：猪圆环病毒2型(PCV2) REP蛋白 间接ELISA 

分 类 号：S852.651[农业科学—基础兽医学]