基于CHO-K1细胞稳定表达的非洲猪瘟病毒CD2v蛋白的间接ELISA抗体检测方法的建立  

Development of an indirect ELISA based on African swine fever virus CD2v protein stably expressed in CHO-K1 cells

作　　者：蒋智勇 蔡汝健 李艳 郭怡德 楚品品 宋帅 勾红潮 李春玲 JIANG Zhi-yong;CAI Ru-jian;LI Yan;GUO Yi-de;CHU Pin-pin;SONG Shuai;GOU Hong-chao;LI Chun-ling(Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture/Guangdong Provincial Key Laboratory of Livestock Disease Prevention/Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China)

机构地区：[1]广东省农业科学院动物卫生研究所广东省畜禽疫病防治研究重点实验室农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640

出　　处：《中国兽医科学》2022年第2期135-142,共8页Chinese Veterinary Science

基　　金：广东省重点领域研发计划项目(2019B020211005);广东省现代农业产业技术体系创新团队项目(2019KJ119);广东省农业科学院院长基金项目(202044)。

摘　　要：为建立非洲猪瘟病毒(ASFV)抗体的间接ELISA检测方法,将构建的GFP基因与CD2v基因串联的重组真核表达质粒pIRES-GFP-CD2v转染CHO-K1细胞,通过嘌呤霉素筛选并结合有限稀释法,筛选稳定表达CD2v的单细胞克隆株。转染细胞经过PCR鉴定后进行扩大培养,收集并纯化细胞培养液,获得目的蛋白,通过Western-blot验证其反应原性。结果表明,ASFV CD2v蛋白在CHO-K1细胞中被成功表达,并能与ASFV抗体阳性血清反应。以纯化的GFP-CD2v重组蛋白为包被抗原建立检测ASFV CD2v蛋白抗体的间接ELISA方法,通过方阵试验对间接ELISA方法进行优化,最终确定抗原最佳包被质量浓度为1.25μg/mL,待检血清最佳稀释度为1∶100,酶标抗体最佳稀释度为1∶50000,以此建立的ASFVCD2v蛋白的间接ELISA方法临界值为0.31;该方法仅与ASFV阳性血清发生特异性反应,而与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒、猪圆环病毒2型及猪流行性腹泻病毒等病毒抗体阳性血清均无交叉反应,表明该方法的特异性较强;用该ELISA方法检测阳性血清敏感性可达1∶1600;批内和批间变异系数均小于10%。结果表明,本试验中建立的间接ELISA方法具有良好的特异性、敏感性和重复性,可用于ASFV抗体的检测。In order to establish an indirect ELISA method for detecting antibodies against African swine fever virus(ASFV),the recombinant eukaryotic expression plasmid p IRES-GFP-CD2v was transfected into Chinese Hamster Ovary(CHO-K1)cells.Then the cell clone stably expressing CD2v protein was obtained by purimycin screening combined with limiting dilution method,and expanded cultivation after being identified by PCR.The recombinant ASFV CD2v protein purified from the cell culture supernatant was identified to be correctly expressed in CHO-K1 by Western-blot and reacted with anti-ASFV sera.Finally,an indirect ELISA method for detecting the anti-CD2v antibodies was established using the purified recombinant CD2v protein as the coating antigen.The critical value of this method was optimized to be 0.31 by the matrix test which determined the optimal reaction conditions as follows:the coating antigen mass concentration was 1.25μg/m L,serum dilution was 1∶100,and the enzyme-labeled antibody dilution was 1∶50000.The ELISA method had good specificity that was specific detecting the ASFV positive serum but had no cross-reaction with other positive sera against major porcine viral pathogens including classical swine fever virus,porcine reproductive and respiratory syndrome virus,foot-and-mouth disease virus,pseudorabies virus,porcine circovirus type 2 and porcine epidemic diarrhea virus.Its sensitivity of detecting the anti-ASFV antibodies was 1∶1600.The coefficients of variation of intra-and interassays were less than 10%.In conclusion,the indirect ELISA established in this study had good specificity,sensitivity and reproducibility,and could be used for the detection of anti-ASFV antibodies.

关 键 词：非洲猪瘟病毒 CD2v CHO-K1细胞 稳定表达 间接ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]