牛病毒性腹泻病毒(BVDV)E0蛋白原核表达及间接ELISA方法的建立
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摘要
牛病毒性腹泻病毒(BVDV)能够引起牛羊的持续感染,临床上很难对其根除,建立该病有效检测方法对净化畜群具有重要意义。本研究构建BVDV E0蛋白基因重组表达载体在大肠杆菌中诱导表达,用重组的E0蛋白为包被抗原,确定抗原最佳包被浓度为5μg/mL,最佳血清稀释比为1∶10,最佳封闭液为5%脱脂奶粉,最佳封闭条件为37℃封闭2 h,酶标二抗稀释度为1∶5000,作用时间为30 min,样品临界值X+2S为0.278。该方法与IDEXX的BVDV间接ELISA试剂盒比较总符合率为86%,与口蹄疫病毒、牛支原体、牛传染性鼻气管炎病毒、牛副结核、牛布鲁氏菌病阳性血清无交叉反应。该检测方法能够用于BVDV抗体水平的临床检测,可为感染病畜的合理淘汰提供实验依据。
Bovine viral diarrhea virus caused persistent infection in cattle and sheep and was hard to eradicate, establishing a effective method to detect the antibody of bovine viral diarrhea virus(BVDV) was significant for cleaning out infected animals. The envelope protein E0 of BVDV was successfully expressed in E.coli expression system. The recombinant and purified E2 protein was coated in ELISA plates as antigen. The optimal working parameters were determined as follows, the antigen concentration was 5μg/mL, the serum dilution was 1∶10,the optimal coating buffer was 5% skimmed milk, the block time was 2 h at 37 ℃,the second-antibody concentration was 1∶5000 for 30 min,the cut-off value was 0.278. Compared with foreign commercial kits, the total coincidence rate was 86%. There was no cross-reaction with the positive sera of M. bovis, FMDV, BPIV-3, IBRV,Mycobacterium paratuberculosis and Brucella abortus. The indirect ELISA method can be applied in BVDV antibody detection for epidemiological investigation and offer experimental evidence for eliminating infected animals.
Bovine viral diarrhea virus caused persistent infection in cattle and sheep and was hard to eradicate, establishing a effective method to detect the antibody of bovine viral diarrhea virus(BVDV) was significant for cleaning out infected animals. The envelope protein E0 of BVDV was successfully expressed in E.coli expression system. The recombinant and purified E2 protein was coated in ELISA plates as antigen. The optimal working parameters were determined as follows, the antigen concentration was 5μg/mL, the serum dilution was 1∶10,the optimal coating buffer was 5% skimmed milk, the block time was 2 h at 37 ℃,the second-antibody concentration was 1∶5000 for 30 min,the cut-off value was 0.278. Compared with foreign commercial kits, the total coincidence rate was 86%. There was no cross-reaction with the positive sera of M. bovis, FMDV, BPIV-3, IBRV,Mycobacterium paratuberculosis and Brucella abortus. The indirect ELISA method can be applied in BVDV antibody detection for epidemiological investigation and offer experimental evidence for eliminating infected animals.
引文
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[5]Lang Q,Wang F,Yin L,et al.Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen[J].Analytical Chemistry,2014,86(5):2767-2774.
[6]Giammarioli M,Ceglie L,Rossi E,et al.Increased genetic diversity of BVDV-1:recent findings and implications thereof[J].Virus Genes,2015,50(1):147.
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[8]Ellis J A,Martin K,Norman G R,et al.Comparison of detection methods for bovine viral diarrhea virus in bovine abortions and neonatal death[J].Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2014,7(4):433.
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[10]刘军,尹惠琼,颜仁和,等.牛病毒性腹泻病毒E0基因重组慢病毒载体构建和鉴定[J].中国预防兽医学报,2015,37(5):357-360.Liu J,Yin H Q,Yan R H,et al.Construction and identification of lentiviral vector for bovine viral diarrhea virus E0 gene[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):357-360.
[11]Pinior B,Firth C L,Richter V,et al.A systematic review of financial and economic assessments of bovine viral diarrhea virus(BVDV)prevention and mitigation activities worldwide[J].Preventive Veterinary Medicine,2017,137(7):77-92.
[12]Lanyon S R,Hill F I,Reichel M P,et al.Bovine viral diarrhoea:pathogenesis and diagnosis[J].Veterinary Journal,2014,199(2):201-209.
[13]李文文,刘情,刘亚刚,等.牛病毒性腹泻病毒间接ELISA方法的建立[J].中国畜牧兽医,2015,42(3):558-562.Li W W,Liu Q,Liu Y G.Establishment of indirect ELISA method of bovine viral diarrhea virus[J].China Animal Husbandry&Veterinary Medicine,2015,42(3):558-562.
[14]Duncan A J,Gunn G J,Humphry R W,et al.Difficulties arising from the variety of testing schemes used for bovine viral diarrhoea virus(BVDV)[J].Veterinary Record,2016,178:123-134.
[15]Sandvik T.Selection and use of laboratory diagnostic assays in BVD control programmes[J].Preventive Veterinary Medicine,2005,72(1):3-16.
[16]Diéguez F J,Cervi o M,Yus E,et al.Bovine viral diarrhea virus(BVDV)genetic diversity in Spain:A review[J].Spanish Journal of Agricultural Research,2017,15(2):23-28.
[2]韩猛立,黄新,钟发刚,等.牛病毒性腹泻病流行现状与防制[J].中国奶牛,2010,12(3):35-38.
[3]Donis R O,Corapi W V,Dubovi E J,et al.Bovine viral diarrhea virus proteins and their antigenic analyses[J].Archives of Virology Supplementum,1991,3(3):29.
[4]Ammari M,Mccarthy F M,Nanduri B,et al.Analysis of bovine viral diarrhea viruses-infected monocytes:identification of cytopathic and non-cytopathic biotype differences[J].Bmc Bioinformatics,2010,11(6):9-17.
[5]Lang Q,Wang F,Yin L,et al.Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen[J].Analytical Chemistry,2014,86(5):2767-2774.
[6]Giammarioli M,Ceglie L,Rossi E,et al.Increased genetic diversity of BVDV-1:recent findings and implications thereof[J].Virus Genes,2015,50(1):147.
[7]Deng M,Ji S,Fei W,et al.Prevalence study and genetic typing of bovine viral diarrhea virus(BVDV)in four bovine species in China[J].Plos One,2015,10(4):121-137.
[8]Ellis J A,Martin K,Norman G R,et al.Comparison of detection methods for bovine viral diarrhea virus in bovine abortions and neonatal death[J].Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2014,7(4):433.
[9]Mayo M A.A summary of taxonomic changes recently approved by ICTV[J].Archives of Virology,2002,147(8):1655-1663.
[10]刘军,尹惠琼,颜仁和,等.牛病毒性腹泻病毒E0基因重组慢病毒载体构建和鉴定[J].中国预防兽医学报,2015,37(5):357-360.Liu J,Yin H Q,Yan R H,et al.Construction and identification of lentiviral vector for bovine viral diarrhea virus E0 gene[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(5):357-360.
[11]Pinior B,Firth C L,Richter V,et al.A systematic review of financial and economic assessments of bovine viral diarrhea virus(BVDV)prevention and mitigation activities worldwide[J].Preventive Veterinary Medicine,2017,137(7):77-92.
[12]Lanyon S R,Hill F I,Reichel M P,et al.Bovine viral diarrhoea:pathogenesis and diagnosis[J].Veterinary Journal,2014,199(2):201-209.
[13]李文文,刘情,刘亚刚,等.牛病毒性腹泻病毒间接ELISA方法的建立[J].中国畜牧兽医,2015,42(3):558-562.Li W W,Liu Q,Liu Y G.Establishment of indirect ELISA method of bovine viral diarrhea virus[J].China Animal Husbandry&Veterinary Medicine,2015,42(3):558-562.
[14]Duncan A J,Gunn G J,Humphry R W,et al.Difficulties arising from the variety of testing schemes used for bovine viral diarrhoea virus(BVDV)[J].Veterinary Record,2016,178:123-134.
[15]Sandvik T.Selection and use of laboratory diagnostic assays in BVD control programmes[J].Preventive Veterinary Medicine,2005,72(1):3-16.
[16]Diéguez F J,Cervi o M,Yus E,et al.Bovine viral diarrhea virus(BVDV)genetic diversity in Spain:A review[J].Spanish Journal of Agricultural Research,2017,15(2):23-28.