猪繁殖与呼吸综合征病毒Nsp4抗体ELISA检测方法的建立
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摘要
克隆、表达了猪繁殖与呼吸综合征病毒DY株(GenBank:JN864948)的Nsp4蛋白,并利用Nsp4作为包被蛋白建立了ELISA检测方法。优化的Nsp4抗体ELISA检测方法的最佳抗原包被浓度为1μg/mL,血清最佳稀释度为1∶40,最佳封闭液为100 mL/L牛血清,酶标二抗的最佳工作稀释度为1∶4 000,最佳显色时间为37℃反应15 min。用建立的ELISA检测方法和IDEXX ELISA检测试剂盒检测血清样品130份,总符合率为93.19%。Nsp4抗体ELISA检测方法的建立,可为PRRSV的抗体监测提供技术支持。
In this study,Nsp4 gene was cloned and expressed from porcine reproductive and respiratory syndrome virus DY strain(GenBank:JN864948),and a Nsp4 antibody ELISA detection method was established by using Nsp4 protein.The optimal antigen package was 1 μg/mL,the best dilution of the serum was 1∶40,the best closed liquid was 100 ml/L bovine serum,the best working dilution of enzyme labeled secondary antibody was 1∶4 000,and the best coloring time at 37℃ was 15 min.For compliance experiment,130 serum samples were detected by IDEXX ELISA Kit,and the total compliance rate was 93.19%.The establishment of Nsp4 antibody ELISA detection method provides a new idea for PRRSV antibody monitoring.
In this study,Nsp4 gene was cloned and expressed from porcine reproductive and respiratory syndrome virus DY strain(GenBank:JN864948),and a Nsp4 antibody ELISA detection method was established by using Nsp4 protein.The optimal antigen package was 1 μg/mL,the best dilution of the serum was 1∶40,the best closed liquid was 100 ml/L bovine serum,the best working dilution of enzyme labeled secondary antibody was 1∶4 000,and the best coloring time at 37℃ was 15 min.For compliance experiment,130 serum samples were detected by IDEXX ELISA Kit,and the total compliance rate was 93.19%.The establishment of Nsp4 antibody ELISA detection method provides a new idea for PRRSV antibody monitoring.
引文
[1] 陈如敬,周伦江,吴学敏,等.猪繁殖与呼吸综合征病毒抗原表位研究进展[J].动物医学进展,2016,37(11):91-93.
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[5] Niran A,Tippawan J.Structural insights into type Ⅰ and type Ⅱ of Nsp4 porcine reproductive and respiratory syndrome virus (Nsp4 PRRSV) by molecular dynamicss imulations [J] .J Mol Graph Model,2017(74):125-134.
[6] Ma Z,Wang Y,Zhao H,et al.Porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3C-like serine protease activity[J].PLoS One,2013,8(7):e69387.
[7] 刘亚丽,丁耀忠,张杰.猪繁殖与呼吸综合征病毒非结构蛋白研究进展[J].中国畜牧兽医,2016,43(8):183-189.
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[9] 李志杰,提金凤,李舫,等.猪繁殖与呼吸综合征病毒GP5蛋白的表达及间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2018,40(1):24-28.
[10] Baker R B,Halbur P G.Use of an experimental model to test the efficacy of planned exposuret live porcine reproductive and respiratory syndrome virus[J].Clin Vac Immunol,2007,14(12):1572-1577.
[11] Xu A T,Zhou Y J,Li G X,et al.Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus[J].Biotechnol Lett,2010,32(12):1905-1910.
[12] Yuan S Z,Zhang N,Xu L,et al.Induction of apoptosis by the nonstructural protein 4 and 10 of porcine reproductive and respiratory syndrome virus[J].PLoS One,2016,11(6):e0156518.
[13] Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hall mark[J].PLoS One,2007,2(6):e526.
[14] Fang Y,Snijder E J.The PRRSV replicase:Exploring the multifunctionality of an intriguing set of nonstructural proteins[J].Virus Res,2010,154(12):61-67.
[15] Li Y,Zhou L,Zhang J,et al.Nsp9 and Nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China [J].PLoS Pathog,2014,10(7):1-18.
[16] Brown E,Lawson S.Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types Ⅰ and Ⅱ [J].Clin Vac Immunol,2009,16(5):628-635.
[2] 王凤雪,刘莹,杨勇,等.高致病性PRRSV JL-04/12株核衣壳蛋白的表达与抗原性分析[J].动物医学进展,2015,36(10):60-64.
[3] Wang F X,Guo L,Yang Y L,et al.Isolation and sequence analysis of highly pathogenic porcine reproductive and respiratory syndrome virus from swine herds in the Jilin province of China[J].Indian J Virol,2013,24(1):90-92.
[4] Dea S,Gagnon C H,Pirzadeh B,et al.Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus:comparison of the North American and European isolates[J].Arch Virol,2000,145(4):659-688.
[5] Niran A,Tippawan J.Structural insights into type Ⅰ and type Ⅱ of Nsp4 porcine reproductive and respiratory syndrome virus (Nsp4 PRRSV) by molecular dynamicss imulations [J] .J Mol Graph Model,2017(74):125-134.
[6] Ma Z,Wang Y,Zhao H,et al.Porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3C-like serine protease activity[J].PLoS One,2013,8(7):e69387.
[7] 刘亚丽,丁耀忠,张杰.猪繁殖与呼吸综合征病毒非结构蛋白研究进展[J].中国畜牧兽医,2016,43(8):183-189.
[8] Liu H L,Fang Y.Nuclear localization of porcine reproductive and respiratory syndrome virus nonstructural protein 4 [J].Nanjing Agric Univ,2011,34(4):89-94.
[9] 李志杰,提金凤,李舫,等.猪繁殖与呼吸综合征病毒GP5蛋白的表达及间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2018,40(1):24-28.
[10] Baker R B,Halbur P G.Use of an experimental model to test the efficacy of planned exposuret live porcine reproductive and respiratory syndrome virus[J].Clin Vac Immunol,2007,14(12):1572-1577.
[11] Xu A T,Zhou Y J,Li G X,et al.Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus[J].Biotechnol Lett,2010,32(12):1905-1910.
[12] Yuan S Z,Zhang N,Xu L,et al.Induction of apoptosis by the nonstructural protein 4 and 10 of porcine reproductive and respiratory syndrome virus[J].PLoS One,2016,11(6):e0156518.
[13] Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hall mark[J].PLoS One,2007,2(6):e526.
[14] Fang Y,Snijder E J.The PRRSV replicase:Exploring the multifunctionality of an intriguing set of nonstructural proteins[J].Virus Res,2010,154(12):61-67.
[15] Li Y,Zhou L,Zhang J,et al.Nsp9 and Nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China [J].PLoS Pathog,2014,10(7):1-18.
[16] Brown E,Lawson S.Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types Ⅰ and Ⅱ [J].Clin Vac Immunol,2009,16(5):628-635.