H3N2亚型SIV血凝蛋白单克隆抗体的制备
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摘要
【目的】制备H3N2亚型猪流感病毒(SIV)的单克隆抗体,为SIV的鉴别诊断奠定基础。【方法】将A/swine/Henan/1/2010(H3N2)猪流感病毒初步浓缩后,免疫6周龄的BALB/c小鼠,取小鼠脾细胞与瘤细胞NS0融合,采用间接ELISA方法筛选杂交瘤细胞,对杂交瘤细胞分泌的单克隆抗体进行Western blot检验和抗原结合活性检测。【结果】经过3次有限稀释法克隆纯化,最终得到1株能稳定分泌单克隆抗体的阳性细胞株,命名为1C10,经检测其腹水抗体效价为1∶51 200。单克隆抗体亚型鉴定结果表明,1C10为IgG1亚型,轻链类型为κ链。Western blot检测结果表明,这株单克隆抗体能与H3N2特异性结合,而且能特异性识别血凝素蛋白(HA),而不与H1N1亚型猪流感病毒、伪狂犬病毒、猪圆环病毒2型、猪细小病毒发生交叉反应。【结论】制备获得1株抗猪流感H3N2病毒HA蛋白的单克隆抗体,可用于猪流感病毒鉴别诊断方法的建立。
【Objective】To identify different swine influenza virus(SIV)and antibodies against SIV,monoclonal antibodies(Mabs)secreted by hybridoma cell lines against SIV H3 subtype were prepared.【Method】Concentrated swine influenza A/swine/Henan/1/2010(H3N2)viruses were injected subcutaneously or intraperitoneally into 6-week-old BALB/c mice with the rate of 0.2mL per mouse according to the immunization protocol.For fusion,spleen cells and partner cells were washed,harvested,and mixed.Hybridomas cells were screened using indirect ELISA method.Then,the monocolonal antibodies from hybridomas were tested by Western blot.【Result】One candidate hybridoma named 1C10 was identified after clone and purification by limiting dilution for three times.The titre of ascitic fluids of 1C10 was up to 1∶51 200.Monoclonal antibody 1C10 was IgG1subtype and its light chain was kappa-type.Western blot showed that the MAb was specifically bounded to SIV H3N2 virus.1C10can be captured by hemagglutinin protein(HA)protein of SIV H3N2 in indirect ELISA and Western blot tests.There was no crossing reaction between 1C10,SIV H1N1 subtype,PRRSV,PCV2,and PPV.【Conclusion】The obtained monoclonal antibody1C10 could be a useful reagent for diagnosing SIV infection and analyzing the epitopes of HA protein of SIV H3N2 virus.
【Objective】To identify different swine influenza virus(SIV)and antibodies against SIV,monoclonal antibodies(Mabs)secreted by hybridoma cell lines against SIV H3 subtype were prepared.【Method】Concentrated swine influenza A/swine/Henan/1/2010(H3N2)viruses were injected subcutaneously or intraperitoneally into 6-week-old BALB/c mice with the rate of 0.2mL per mouse according to the immunization protocol.For fusion,spleen cells and partner cells were washed,harvested,and mixed.Hybridomas cells were screened using indirect ELISA method.Then,the monocolonal antibodies from hybridomas were tested by Western blot.【Result】One candidate hybridoma named 1C10 was identified after clone and purification by limiting dilution for three times.The titre of ascitic fluids of 1C10 was up to 1∶51 200.Monoclonal antibody 1C10 was IgG1subtype and its light chain was kappa-type.Western blot showed that the MAb was specifically bounded to SIV H3N2 virus.1C10can be captured by hemagglutinin protein(HA)protein of SIV H3N2 in indirect ELISA and Western blot tests.There was no crossing reaction between 1C10,SIV H1N1 subtype,PRRSV,PCV2,and PPV.【Conclusion】The obtained monoclonal antibody1C10 could be a useful reagent for diagnosing SIV infection and analyzing the epitopes of HA protein of SIV H3N2 virus.
引文
[1]Ma W,Gramer M,Rossow K,et al.Isolation and genetic characterization of new reassortant H3N1swine influenza virus from pigs in the Midwestern United States[J].J Virol,2006,80:5092-5096.
[2]陈敬贤.2010-2011年流感病毒流行状况回顾[J].中华结核和呼吸杂志,2012,35(1):61-62.Chen J X.Review of the 2010-2011influenza[J].Chinese Journal of Tuberculosis and Respiratory Diseases,2012,35(1):61-62.(in Chinese)
[3]李海燕,于康震,杨焕良,等.中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J].中国预防兽医学报,2004,26(1):1-6.Li H Y,Yu K Z,Yang H L,et al.Isolation and identification of Chinese swine H5N1and H9N2subtype of influenza virus[J].Chinese Journal of Preventive Veterinary Medicine,2004,26(1):1-6.(in Chinese)
[4]周伦江,王隆柏,俞玉鸿,等.人源H3N2亚型猪流感重组病毒的分离鉴定及全基因序列分析[J].中国预防兽医学报,2011,33(9):675-680.Zhou L J,Wang L B,Yu Y H,et al.Isolation and identification of human H3N2subtype swine influenza viruses and sequence analysis of recombinant genome[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(9):675-680.(in Chinese)
[5]Scotch M,Mei C.Phylogeography of swine influenza H3N2in the United States:Translational public health for zoonotic disease surveillance[J].Infection,Genetics&Evolution,2013,13(1):224-229.
[6]张云.河南省H1N2亚型猪流感病毒分离株基因组演化特征和生物学特性研究[D].郑州:河南农业大学,2012.Zhang Y.Research and biological characteristics of Henan H1N2subtype swine influenza virus isolate genomic evolution[D].Zhengzhou:Henan Agricultural University,2012.(in Chinese)
[7]杨建民,秦卓明.禽脑脊髓炎病毒单克隆抗体的制备及鉴定[J].畜牧兽医学报,2003,34(2):191-194.Yang J M,Qin Z M.Preparation and characterization of avian encephalomyelitis virus monoclonal antibody[J].Acta Veterinaria et Zootechnica Sinica,2003,34(2):191-194.(in Chinese)
[8]Crowther J R.The ELISA guidebook[M].2nd ed.USA:Humana Press,2009:79-111.
[9]陈艳,辛晓光,杨焕良,等.猪流感抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2007,29(4):308-311.Chen Y,Xin X G,Yang H L,et al.Swine flu establish an indirect ELISA detection method[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(4):308-311.(in Chinese)
[10]郭利敏,乔传玲,陈艳,等.利用真核表达的猪流感病毒NP蛋白制备其特异性的单克隆抗体[J].畜牧兽医学报,2010,32(6):460-463.Guo L M,Qiao C L,Chen Y,et al.The use of eukaryotic expression swine influenza virus NP protein specificity monoclonal antibody preparation[J].Acta Veterinaria et Zootechnica Sinica,2010,32(6):460-463.(in Chinese)
[11]李慧昕,刘胜旺,韩宗玺,等.鸭肠炎病毒VP5蛋白单克隆抗体的制备及鉴定[J].中国兽医杂志,2013,29(1):9-12.Li H X,Liu S W,Han Z X,et al.Preparation and characterization of duck enteritis virus VP5protein monoclonal antibodies[J].Chinese Journal of Veterinary Medicine,2013,29(1):9-12.(in Chinese)
[12]汪招雄,何启盖,黄红亮,等.抗伪狂犬病毒Ea株单克隆抗体的制备及鉴定[J].华中农业大学学报,2005,24(3):222-225.Wang Z X,He Q G,Huang H L,et al.Preparation and characterization of anti-pseudorabies virus Ea monoclonal antibodies[J].Huazhong Agricultural University,2005,24(3):222-225.(in Chinese)
[13]肖成蕊,宋战昀,杨立英,等.H3N8亚型马流感病毒单克隆抗体的制备及鉴定[J].动物医学进展,2011,32(6):1-4.Xiao C R,Song Z Y,Yang L Y,et al.Preparation and characterization of equine influenza virus subtype H3N8monoclonal antibodies[J].Progress in Veterinary Medicine,2011,32(6):1-4.(in Chinese)
[14]李引乾,郝志明,段清学,等.鸡胚尿囊液对小鼠免疫功能与抗应激作用的影响[J].中国兽医科技,2003,33(11):46-48.Li Y Q,Hao Z M,Duan Q X,et al.Allantoic fluid impact on immune function and anti-stress effects in mice[J].Chinese Journal of Veterinary Science and Technology,2003,33(11):46-48.(in Chinese)
[2]陈敬贤.2010-2011年流感病毒流行状况回顾[J].中华结核和呼吸杂志,2012,35(1):61-62.Chen J X.Review of the 2010-2011influenza[J].Chinese Journal of Tuberculosis and Respiratory Diseases,2012,35(1):61-62.(in Chinese)
[3]李海燕,于康震,杨焕良,等.中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J].中国预防兽医学报,2004,26(1):1-6.Li H Y,Yu K Z,Yang H L,et al.Isolation and identification of Chinese swine H5N1and H9N2subtype of influenza virus[J].Chinese Journal of Preventive Veterinary Medicine,2004,26(1):1-6.(in Chinese)
[4]周伦江,王隆柏,俞玉鸿,等.人源H3N2亚型猪流感重组病毒的分离鉴定及全基因序列分析[J].中国预防兽医学报,2011,33(9):675-680.Zhou L J,Wang L B,Yu Y H,et al.Isolation and identification of human H3N2subtype swine influenza viruses and sequence analysis of recombinant genome[J].Chinese Journal of Preventive Veterinary Medicine,2011,33(9):675-680.(in Chinese)
[5]Scotch M,Mei C.Phylogeography of swine influenza H3N2in the United States:Translational public health for zoonotic disease surveillance[J].Infection,Genetics&Evolution,2013,13(1):224-229.
[6]张云.河南省H1N2亚型猪流感病毒分离株基因组演化特征和生物学特性研究[D].郑州:河南农业大学,2012.Zhang Y.Research and biological characteristics of Henan H1N2subtype swine influenza virus isolate genomic evolution[D].Zhengzhou:Henan Agricultural University,2012.(in Chinese)
[7]杨建民,秦卓明.禽脑脊髓炎病毒单克隆抗体的制备及鉴定[J].畜牧兽医学报,2003,34(2):191-194.Yang J M,Qin Z M.Preparation and characterization of avian encephalomyelitis virus monoclonal antibody[J].Acta Veterinaria et Zootechnica Sinica,2003,34(2):191-194.(in Chinese)
[8]Crowther J R.The ELISA guidebook[M].2nd ed.USA:Humana Press,2009:79-111.
[9]陈艳,辛晓光,杨焕良,等.猪流感抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2007,29(4):308-311.Chen Y,Xin X G,Yang H L,et al.Swine flu establish an indirect ELISA detection method[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(4):308-311.(in Chinese)
[10]郭利敏,乔传玲,陈艳,等.利用真核表达的猪流感病毒NP蛋白制备其特异性的单克隆抗体[J].畜牧兽医学报,2010,32(6):460-463.Guo L M,Qiao C L,Chen Y,et al.The use of eukaryotic expression swine influenza virus NP protein specificity monoclonal antibody preparation[J].Acta Veterinaria et Zootechnica Sinica,2010,32(6):460-463.(in Chinese)
[11]李慧昕,刘胜旺,韩宗玺,等.鸭肠炎病毒VP5蛋白单克隆抗体的制备及鉴定[J].中国兽医杂志,2013,29(1):9-12.Li H X,Liu S W,Han Z X,et al.Preparation and characterization of duck enteritis virus VP5protein monoclonal antibodies[J].Chinese Journal of Veterinary Medicine,2013,29(1):9-12.(in Chinese)
[12]汪招雄,何启盖,黄红亮,等.抗伪狂犬病毒Ea株单克隆抗体的制备及鉴定[J].华中农业大学学报,2005,24(3):222-225.Wang Z X,He Q G,Huang H L,et al.Preparation and characterization of anti-pseudorabies virus Ea monoclonal antibodies[J].Huazhong Agricultural University,2005,24(3):222-225.(in Chinese)
[13]肖成蕊,宋战昀,杨立英,等.H3N8亚型马流感病毒单克隆抗体的制备及鉴定[J].动物医学进展,2011,32(6):1-4.Xiao C R,Song Z Y,Yang L Y,et al.Preparation and characterization of equine influenza virus subtype H3N8monoclonal antibodies[J].Progress in Veterinary Medicine,2011,32(6):1-4.(in Chinese)
[14]李引乾,郝志明,段清学,等.鸡胚尿囊液对小鼠免疫功能与抗应激作用的影响[J].中国兽医科技,2003,33(11):46-48.Li Y Q,Hao Z M,Duan Q X,et al.Allantoic fluid impact on immune function and anti-stress effects in mice[J].Chinese Journal of Veterinary Science and Technology,2003,33(11):46-48.(in Chinese)