鸭圆环病毒ORF3蛋白多克隆抗体的制备与应用
|
摘要
鸭圆环病毒(DuCV)感染引起淋巴细胞凋亡,其ORF3蛋白是唯一具有凋亡活性的病毒蛋白,但目前ORF3特异性抗体的缺乏限制了其生物学功能的深入研究。DuCV包含基因I型和基因II型两种基因型,利用软件分析两基因型ORF3同源性,选取保守性肽段进行多肽合成,并与钥孔血蓝蛋白(KLH)偶联,多次加强免疫兔子,采集血清并纯化IgG后制备ORF3多克隆抗体。ELISA检测发现此抗体能特异性与ORF3保守肽段反应,并具有较高的效价,间接免疫荧光(IFA)显示此抗体能特异性识别两基因型ORF3蛋白,但并不能用于western blot检测,说明此多抗主要识别ORF3空间构象的表位。总之,我们成功制备了特异性识别两种基因型DuCV ORF3蛋白的抗体,为今后深入探究DuCV ORF3蛋白的生物学功能提供了良好的研究工具。
引文
[1] Hattermann, K., Schmitt, C., Soike, D., Mankertz,A.Cloning and sequencing of Duck circovirus(DuCV)[J].Archives of virology,2003,148:2471-2480.
[2] Hong Y T, Kang M, Jang H K. Pathogenesis of duck circovirus genotype 1 in experimentally infected Pekin ducks[J].Poultry Science, 2018, 97:3050-3057.
[3]刘少宁,杨金保,张兴晓,等.鸭圆环病毒FJ0601株Rep蛋白的原核表达及其抗血清制备[J].中国兽医学报, 2010(1):10-12.
[4] Xiang Q W, Wang X, Xie Z J, et al. ORF3 of duck circovirus:A novel protein with apoptotic activity[J].Veterinary Microbiology,2012,159(1-2):251-256.
[5] Wang D, Xie X, Zhang D, et al. Detection of duck circovirus in China:A proposal on genotype classification[J].Veterinary Microbiology,2011,147(3-4):410-415.
[6]魏宗,邹金峰,雷战,等.鸭圆环病毒ORF3基因的克隆及序列分析[J].中国预防兽医学报,2011,33(9):746-748.
[7] Wu Z C, Zhang R H, Li Y M, et al. C-terminal 20residues of ORF3 protein of duck circovirus genotype2 regulates the nuclear localization and inhibits apoptotic activity of ORF3 protein[J]. Veterinary Microbiology, 2018, 214:21-27.
[8] Banda A,Galloway-Haskins R I,Schat S K A. Genetic Analysis of a Duck Circovirus Detected in Commercial Pekin Ducks in New York(Análisis filogenético de un circovirus de pato detectado en patos pequineses comerciales en Nueva York)[J].Avian Diseases, 2007, 51(1):90-95.
[9] Zhang X, Jiang S, Wu J, et al. An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China[J]. Veterinary Microbiology, 2009, 133(3):252-256.
[10] Cha S Y, Kang M, Cho J G, et al. Genetic analysis of duck circovirus in Pekin ducks from South Korea[J].Poultry Science, 2013, 92(11):2886-2891.
[11] Cha S Y,Song E T, Kang M, et al. Prevalence of Duck Circovirus Infection of Subclinical Pekin Ducks in South Korea[J].Journal of Veterinary Medical Science, 2014, 76(4):597-599.
[12] Wei L, Zhu S, Wang J, et al. Regulatory role of ASK1 in porcine circovirus type 2-induced apoptosis[J]. Virology, 2013, 447(1-2):285-291.
[13] Jeurissen S H M, Wagenaar F, Pol J M A, et al.Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and cell lines after in vitro infection[J]. Journal of Virology, 1993, 66(12):7383-7388.
[14] Todd D.Circoviruses:Immunosuppressive threats to avian species:A review[J]. Avian Pathology.
[2] Hong Y T, Kang M, Jang H K. Pathogenesis of duck circovirus genotype 1 in experimentally infected Pekin ducks[J].Poultry Science, 2018, 97:3050-3057.
[3]刘少宁,杨金保,张兴晓,等.鸭圆环病毒FJ0601株Rep蛋白的原核表达及其抗血清制备[J].中国兽医学报, 2010(1):10-12.
[4] Xiang Q W, Wang X, Xie Z J, et al. ORF3 of duck circovirus:A novel protein with apoptotic activity[J].Veterinary Microbiology,2012,159(1-2):251-256.
[5] Wang D, Xie X, Zhang D, et al. Detection of duck circovirus in China:A proposal on genotype classification[J].Veterinary Microbiology,2011,147(3-4):410-415.
[6]魏宗,邹金峰,雷战,等.鸭圆环病毒ORF3基因的克隆及序列分析[J].中国预防兽医学报,2011,33(9):746-748.
[7] Wu Z C, Zhang R H, Li Y M, et al. C-terminal 20residues of ORF3 protein of duck circovirus genotype2 regulates the nuclear localization and inhibits apoptotic activity of ORF3 protein[J]. Veterinary Microbiology, 2018, 214:21-27.
[8] Banda A,Galloway-Haskins R I,Schat S K A. Genetic Analysis of a Duck Circovirus Detected in Commercial Pekin Ducks in New York(Análisis filogenético de un circovirus de pato detectado en patos pequineses comerciales en Nueva York)[J].Avian Diseases, 2007, 51(1):90-95.
[9] Zhang X, Jiang S, Wu J, et al. An investigation of duck circovirus and co-infection in Cherry Valley ducks in Shandong Province, China[J]. Veterinary Microbiology, 2009, 133(3):252-256.
[10] Cha S Y, Kang M, Cho J G, et al. Genetic analysis of duck circovirus in Pekin ducks from South Korea[J].Poultry Science, 2013, 92(11):2886-2891.
[11] Cha S Y,Song E T, Kang M, et al. Prevalence of Duck Circovirus Infection of Subclinical Pekin Ducks in South Korea[J].Journal of Veterinary Medical Science, 2014, 76(4):597-599.
[12] Wei L, Zhu S, Wang J, et al. Regulatory role of ASK1 in porcine circovirus type 2-induced apoptosis[J]. Virology, 2013, 447(1-2):285-291.
[13] Jeurissen S H M, Wagenaar F, Pol J M A, et al.Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and cell lines after in vitro infection[J]. Journal of Virology, 1993, 66(12):7383-7388.
[14] Todd D.Circoviruses:Immunosuppressive threats to avian species:A review[J]. Avian Pathology.