猪瘟病毒结构蛋白E0抗体间接ELISA检测方法的建立及优化

郭东光; 陈明艳; 朱艳平; 李鹏; 岳锋; 崔芳微; 李文明; 王金海; 王选年

doi:10.15933/j.cnki.1004-3268.2019.11.021

河南农业科学 >

2019 , Vol. 48 >Issue 11: 151 - 156

DOI: https://doi.org/10.15933/j.cnki.1004-3268.2019.11.021

畜牧·兽医

猪瘟病毒结构蛋白E0抗体间接ELISA检测方法的建立及优化

郭东光 ,
陈明艳 ,
朱艳平 ,
李鹏 ,
岳锋 ,
崔芳微 ,
李文明 ,
王金海 ,
王选年

展开

(1.新乡学院生命科学技术学院，河南新乡 453003； 2.郑州大学生命科学技术学院，河南郑州 450000)

郭东光（1987-），男，河南淮阳人，讲师，博士，主要从事动物分子免疫学研究。E-mail：gdg2011@163.com

收稿日期: 2019-06-15

网络出版日期: 2019-11-15

基金资助

河南省科技攻关项目（192102110066）；河南省高等学校重点科研项目(19A230009)；新乡学院博士科研启动经费项目（1366020120）

收起

Establishment and Optimization of Indirect ELISA for Detection of Structural Protein E0 Antibody of Classical Swine Fever Virus

Expand

( 1.School of Life Science,Xinxiang University,Xinxiang 453003,China; 2.School of Life Science,Zhengzhou University,Zhengzhou 450000,China)

Received date: 2019-06-15

Online published: 2019-11-15

Fold

摘要

为建立一种经济、可靠的猪瘟病毒（CSFV）血清抗体检测方法，采用间接ELISA（E0-ELISA）方法，以在大肠杆菌（Escherichia coli）中表达的重组蛋白E0为包被原，优化反应条件。结果显示，优化后的E0-ELISA在包被原1.0 μg/mL质量浓度下37 ℃包被1 h效果最佳，最佳封闭条件为5％脱脂奶粉溶液37 ℃作用2 h，待检血清以1∶100稀释后37 ℃作用60 min效果最佳，二抗以1∶5 000稀释后37 ℃孵育45 min效果最佳。当OD₄₅₀<0.370时，判断为猪血清CSFV抗体阴性；批内和批间重复试验变异系数分别为1.99%~7.69%和2.16%~9.23%，表明该方法具有良好的稳定性；E0-ELISA检测猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪细小病毒(PPV)阳性血清时结果均为阴性；当CSFV阳性血清稀释至1∶640时结果仍为阳性，表明该检测方法具有良好的敏感性；与IDEXX CSFV抗体检测试剂盒相比，E0-ELISA检测符合率为92.00%，敏感性为97.86%，特异性为78.33%。综上，成功建立一种经济、稳定、特异性好和敏感性高的CSFV血清抗体间接ELISA检测方法。

关键词： 猪瘟病毒; 结构蛋白E0; 抗体检测; 间接ELISA

本文引用格式

郭东光 , 陈明艳 , 朱艳平 , 李鹏 , 岳锋 , 崔芳微 , 李文明 , 王金海 , 王选年 . 猪瘟病毒结构蛋白E0抗体间接ELISA检测方法的建立及优化[J]. 河南农业科学, 2019 , 48(11) : 151 -156 . DOI: 10.15933/j.cnki.1004-3268.2019.11.021

Abstract

In order to establish an economical and reliable method for the detection of serum antibody against classical swine fever virus(CSFV),the recombinant protein E0 expressed in Escherichia coli was used as the coating agent，an indirect ELISA(E0-ELISA) was developed for the detection of serum antibody against CSFV.The results showed that the optimized E0-ELISA best effect was gained when the E0 protein was coated at 37℃ for 1 h and the protein mass concentration at 1.0 μg/mL.The best blocking condition was 5% skimmed milk powder solution at 37℃for 2 h.The best effect was obtained when the serum to be tested was diluted at 1∶ 100 and incubated at 37 ℃ for 60 min,and when the second antibody was diluted at 1∶ 5 000 and incubated at 37℃ for 45 min.When OD450＜0.370,it was negative for CSFV antibody.The variation coefficients of intra-batch and inter-batch repeated tests were 1.99%—7.69% and 2.16%—9.23%,respectively,which showed a good stability for the indirect ELISA.The positive serum of PＲＲSV,PＲV,PCV and PPV tested by E0-ELISA showed the negative results.It was still positive when the positive serum CSFV was diluted to 1∶ 640,showing that E0-ELISA had a good sensitivity.Compared with IDEXX CSFV antibody detection kit，the E0-ELISA coincidence rate,sensitivity and specificity were 92.00%,97.86% and 78.33%,respectively.All the results indicated that an economical,stable,specific and sensitive indirect ELISA for detection of CSFV antibodies was successfully established．

Key words： CSFV; Structural protein E0; Antibody detection; Indirect ELISA

参考文献

［1］HAUSE B M,COLLIN E A,PEDDIREDDI L E,et al.Discovery of a novel putative atypical porcine pestivirus in pigs in the USA［J］.Journal of General Virology,2015,96(10):2994-2998.

［2］BROWN V R,BEVINS S N.A Review of classical swine fever virus and routes of introduction into the united states and the potential for virus establishment［J］.Front Vet Sci,2018,5:31-37.

［3］GORAYA M U,ZIAGHUM F,CHEN S,et al.Role of innate immunity in pathophysiology of classical swine fever virus infection［J］.Microb Pathog,2018,119:248-254.

［4］孙亚宁，王丽，王栋，等.猪瘟抗体检测试纸研发及制备工艺的优化［J］.西北农业学报,2019,28(2):169-175.

［5］王春花，孙元，仇华吉.新型猪瘟疫苗研究进展［J］.生物工程学报，2013,29(7):880-890.

［6］PARK Y,AN D J,CHOE S,et al.Development of recombinant proteinbased vaccine against classical swine fver virus in pigs using transgenic nicotiana benthamiana［J］.Front Plant Sci,2019,10:624-632.

［7］LI H,GAO R,ZHANG Y.A promising trigene recombinant human adenovirus vaccine against classical swine fever virus［J］.Viral Immunol,2016,29(4):244-251.

［8］DONG X N,CHEN Y H.Marker vaccine strategies and candidate CSFV marker vaccines［J］.Vaccine,2005,25(4):205-230.

［9］VAN OIRSCHOT J T.Vaccinology of classical swine fever:From lab to field［J］.Vet Microbiol,2003，96(4)：367-384.

［10］ANDREA A,MLLER M,HOFMANN M A.Two newly developed Ernsbased ELISAs allow the differentiation of classical swine fever virusinfected from markervaccinated animals and the discrimination of pestivirus antibodies［J］.Vet Microbiol,2013,106(3):274-285.

［11］MEYER D,FRITSCHE S,LUO Y,et al.Detection of Ernsspecific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals［J］.Transbound Emerg Dis,2017,64(6):2013-2022.

［12］郭东光，朱艳平，孙国鹏，等.猪瘟病毒E0蛋白的原核表达及鉴定［J］.动物医学进展,2014,35(5):31-35.

［13］陈振海，王琴，范学政，等.猪瘟病毒E0蛋白的原核表达及其间接ELISA检测方法的建立［J］.中国病毒学,2005,20(2):135-139.

［14］XU Y G,CUI L C,TIAN C Y,et al.Immunogenicity of recombinant classic swine fever virus CD8+ T lymphocyte epitope and porcine parvovirus VP2 antigen coexpressed by lactobacillus casei in swine via oral vaccination［J］.Clin Vaccine Immunol November,2011,18(11):1979-1986.

［15］LIM S I,CHOE S,KIM K S,et al.Assessment of the efficacy of an attenuated live marker classical swine fever vaccine(Flc-LOM BErns) in pregnant sows［J］.Vaccine,2019,37(27):3598-3604.

［16］JI S,LUO Y,ZHANG T,et al.An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells［J］.Arch Virol,2018,163(7):1831-1839.

［17］PANYASING Y,KEDKOVID R,THANAWONGNUWECH R,et al.Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities［J］.Vet Microbiol,2018,111(6):72-78.

［18］PANNHORST K,FROHLICH A,STAUBACH C,et al.Evaluation of an Erns based enzymelinked immunosorbent assay to distinguish classical swine fever virusinfected pigs from pigs vaccinated with CP7_E2alf［J］.J Vet Diagn Invest,2015,27(4):449-460.

［19］袁莉，杨顺利，尚佑军，等.猪瘟病毒结构蛋白抗体间接ELISA 检测方法的建立及评价［J］.西北农业学报,2018,27(9):1273-1279.

［20］吴素丽，孙惠玲，钱永华.猪瘟病毒E0蛋白的高效表达及其间接ELISA检测方法的建立［J］.动物医学进展,2009,30(8):13-18.

［21］李鹏，张彦明，张志，等.猪瘟流行毒株E0蛋白的原核表达及其间接ELISA方法的建立［J］.西北农林科技大学学报(自然科学版),2008,35(10):24-28.

［22］蔺辉星，周红，童泽鑫，等.猪瘟病毒血清抗体间接 ELISA 检测方法的建立［J］.中国兽医科学,2017,47(1)：9-15.

Options

文章导航

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献