抗博尔纳病病毒磷蛋白单克隆抗体的制备及鉴定
|
摘要
目的以重组的博尔纳病病毒(BDV)磷蛋白(p24)为免疫原制备单克隆抗体,并鉴定其特异性。方法以纯化后的重组BDVp24免疫小鼠,将骨髓瘤细胞与其脾细胞进行融合,经ELISA法筛选出分泌抗p24单抗的细胞株,并对其进行克隆与亚克隆培养,将最终获得的其中2株细胞株分别注入小鼠腹腔制备单抗,经亲和纯化法纯化后,采用ELISA法测定效价,利用蛋白免疫印迹和免疫荧光鉴定其特异性。结果建立了2株稳定分泌抗p24单抗的杂交瘤细胞株,抗体亚类均为IgG1型。纯化后的腹水抗p24单抗纯度为98%和93%,ELISA效价在1∶81 000以上,该抗体均能识别重组p24蛋白和BDV感染人类少突胶质细胞(Oligodendroglia cell,OL细胞)所表达的p24蛋白。结论成功制备了灵敏性高、特异性好的抗BDVp24单抗,为博尔纳病病毒诊断方法的研制及感染机理的研讨提供依据。
In order to prepare the monoclonal antibodies(MAbs)by the recombinant phosphoprotein(p24)of Borne disease virus(BDV)as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay(IFA)for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98% and 93%,and a titer of 1∶81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells(OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
In order to prepare the monoclonal antibodies(MAbs)by the recombinant phosphoprotein(p24)of Borne disease virus(BDV)as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay(IFA)for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98% and 93%,and a titer of 1∶81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells(OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
引文
[1]Kinnunen PM,Palva A,Vaheri A,et al.Epidemiology and host spectrum of Borna disease virus infections[J].J Gen Virol,2013,94(Pt 2):247-262.DOI:10.1099/vir.0.046961-0
[2]Priestnall SL,Sch9niger S,Ivens PA,et al.Borna disease virus infection of a horse in Great Britain[J].Vet Rec,2011,168(14):380b.DOI:10.1136/vr.c6405
[3]Payne SL,Delnatte P,Guo J,et al.Birds and bornaviruses[J].Anim Health Res Rev,2012,13(2):145-156.DOI:10.1017/S1466252312000205
[4]Bode L,Dürrwald R,Rantam FA,et al.First isolates of infectious human Borna disease virus from patients with mood disorders[J].Mol Psychiatry,1996,1(3):200-212.
[5]Nakamura Y,Takahashi H,Shoya Y,et al.Isolation of Borna disease virus from human brain tissue[J].J Virol,2000,74(10):4601-4611.
[6]Guo ZY,Xu P,Yao NY.Detection of the p24nucleotide fragment of Borna disease virus from blood and cerebrospinal fluid in patients with viral encephalitis[J].Chin J Zoonoses,2008,(03):193-197.DOI:10.3969/j.issn.1002-2694.2008.03.002(in Chinese)郭振元,徐平,姚能云.病毒性脑炎患者血液和脑脊液博尔纳病病毒p24核苷酸片段的检测[J].中国人兽共患病学报,2008,(03):193-197.
[7]Zhu D,Xie P,Zeng ZL.Borne disease virus infection and mental behavior abnormalities[J].Chin J Zoonoses,2008,(12):1165-1167.(in Chinese)朱丹,谢鹏,曾志磊.博尔纳病病毒感染与精神行为异常[J].中国人兽共患病学报,2008,(12):1165-1167.
[8]Yang LL,Lei YH,Xu P.Borna disease virus p24in brain tissue of bat infect in Zunyi,Guizhou Province,China[J].Chin J Zoonoses,2016,32(2):156-159.DOI:10.3969/j.issn.1002-2694.2016.02.011(in Chinese)杨利玲,雷以会,徐平.贵州遵义地区蝙蝠脑组织BDV-P24基因片段的检测[J].中国人兽共患病学报.2016,32(2):156-159.DOI:10.3969/j.issn.1002-2694.2016.02.011
[9]Brnic D,Stevanovic V,Cochet M,et al.Borna disease virus infects human neural progenitor cells and impairs neurogenesis[J].J Virol,2012,86(5):2512-2522.DOI:10.1128/JVI.05663-11
[10]Zhang L.A preliminary study on functions,mechanisms of human EBLN1gene and ITS role in major depressive disorder[D].Chongqing:Chongqing Medical University.2014.(in Chinese)张亮.人类EBLN1基因生物学功能、作用机制及其与抑郁症的关系初探[D].重庆:重庆医科大学,2014.
[11]Herzog S,Pfeuffer I,Haberzettl K,et al.Molecular characterization of Borna disease virus from naturally infected animals and possible links to human disorders[J].Arch Virol Suppl,1997,13:183-190.
[12]Xu MM,Zhang YY,Zhan QL,et al.Expression and cellular localization of Borna disease virus P24in different periods after transfection[J].J Third Military Medical Univ,2010,(06):533-536.DOI:10.16016/j.1000-5404.2010.06.009(in Chinese)徐鸣明,张英英,展群岭,等.博尔纳病毒P24转染后不同时期核酸和蛋白的表达及细胞定位[J].第三军医大学学报,2010,(06):533-536.DOI:10.16016/j.1000-5404.2010.06.009
[13]de la Torre JC.Molecular biology of borna disease virus:prototype of a new group of animal viruses[J].J Virol,1994,68(12):7669-7675.
[14]Liu S,Xu P.Laboratory diagnosis of Borne disease[J].Chin J Pract Med,2013,29(15):2437-2439.DOI:10.3969/j.issn.1006-5725.2013.15.009(in Chinese)刘赛,徐平.博尔纳病的实验室诊断方法[J].实用医学杂志.2013,29(15):2437-2439.
[15]Yang QF,Xu P.Research progress of Borne disease virus[J].Chin J Clin Med,2015:165-166.DOI:10.15887/j.cnki.13-1389/r.2015.21.093(in Chinese)杨全凤,徐平.博尔纳病病毒的研究进展[J].临床合理用药杂志,2015:165-166.DOI:10.15887/j.cnki.13-1389/r.2015.21.093
[16]Ma LH,Huang ZR,Zhang L,et al.Preparation and identification of polyclonal antibody against phosphoprotein of Borna disease virus[J].Chin J Biologicals,2013,(02):244-247.DOI:10.13200/j.cjb.2013.02.105.malh.020(in Chinese)马丽华,黄荣忠,张亮,等.抗博尔纳病病毒磷蛋白多克隆抗体的制备及鉴定[J].中国生物制品学杂志,2013,(02):244-247.DOI:10.13200/j.cjb.2013.02.105.malh.020
[2]Priestnall SL,Sch9niger S,Ivens PA,et al.Borna disease virus infection of a horse in Great Britain[J].Vet Rec,2011,168(14):380b.DOI:10.1136/vr.c6405
[3]Payne SL,Delnatte P,Guo J,et al.Birds and bornaviruses[J].Anim Health Res Rev,2012,13(2):145-156.DOI:10.1017/S1466252312000205
[4]Bode L,Dürrwald R,Rantam FA,et al.First isolates of infectious human Borna disease virus from patients with mood disorders[J].Mol Psychiatry,1996,1(3):200-212.
[5]Nakamura Y,Takahashi H,Shoya Y,et al.Isolation of Borna disease virus from human brain tissue[J].J Virol,2000,74(10):4601-4611.
[6]Guo ZY,Xu P,Yao NY.Detection of the p24nucleotide fragment of Borna disease virus from blood and cerebrospinal fluid in patients with viral encephalitis[J].Chin J Zoonoses,2008,(03):193-197.DOI:10.3969/j.issn.1002-2694.2008.03.002(in Chinese)郭振元,徐平,姚能云.病毒性脑炎患者血液和脑脊液博尔纳病病毒p24核苷酸片段的检测[J].中国人兽共患病学报,2008,(03):193-197.
[7]Zhu D,Xie P,Zeng ZL.Borne disease virus infection and mental behavior abnormalities[J].Chin J Zoonoses,2008,(12):1165-1167.(in Chinese)朱丹,谢鹏,曾志磊.博尔纳病病毒感染与精神行为异常[J].中国人兽共患病学报,2008,(12):1165-1167.
[8]Yang LL,Lei YH,Xu P.Borna disease virus p24in brain tissue of bat infect in Zunyi,Guizhou Province,China[J].Chin J Zoonoses,2016,32(2):156-159.DOI:10.3969/j.issn.1002-2694.2016.02.011(in Chinese)杨利玲,雷以会,徐平.贵州遵义地区蝙蝠脑组织BDV-P24基因片段的检测[J].中国人兽共患病学报.2016,32(2):156-159.DOI:10.3969/j.issn.1002-2694.2016.02.011
[9]Brnic D,Stevanovic V,Cochet M,et al.Borna disease virus infects human neural progenitor cells and impairs neurogenesis[J].J Virol,2012,86(5):2512-2522.DOI:10.1128/JVI.05663-11
[10]Zhang L.A preliminary study on functions,mechanisms of human EBLN1gene and ITS role in major depressive disorder[D].Chongqing:Chongqing Medical University.2014.(in Chinese)张亮.人类EBLN1基因生物学功能、作用机制及其与抑郁症的关系初探[D].重庆:重庆医科大学,2014.
[11]Herzog S,Pfeuffer I,Haberzettl K,et al.Molecular characterization of Borna disease virus from naturally infected animals and possible links to human disorders[J].Arch Virol Suppl,1997,13:183-190.
[12]Xu MM,Zhang YY,Zhan QL,et al.Expression and cellular localization of Borna disease virus P24in different periods after transfection[J].J Third Military Medical Univ,2010,(06):533-536.DOI:10.16016/j.1000-5404.2010.06.009(in Chinese)徐鸣明,张英英,展群岭,等.博尔纳病毒P24转染后不同时期核酸和蛋白的表达及细胞定位[J].第三军医大学学报,2010,(06):533-536.DOI:10.16016/j.1000-5404.2010.06.009
[13]de la Torre JC.Molecular biology of borna disease virus:prototype of a new group of animal viruses[J].J Virol,1994,68(12):7669-7675.
[14]Liu S,Xu P.Laboratory diagnosis of Borne disease[J].Chin J Pract Med,2013,29(15):2437-2439.DOI:10.3969/j.issn.1006-5725.2013.15.009(in Chinese)刘赛,徐平.博尔纳病的实验室诊断方法[J].实用医学杂志.2013,29(15):2437-2439.
[15]Yang QF,Xu P.Research progress of Borne disease virus[J].Chin J Clin Med,2015:165-166.DOI:10.15887/j.cnki.13-1389/r.2015.21.093(in Chinese)杨全凤,徐平.博尔纳病病毒的研究进展[J].临床合理用药杂志,2015:165-166.DOI:10.15887/j.cnki.13-1389/r.2015.21.093
[16]Ma LH,Huang ZR,Zhang L,et al.Preparation and identification of polyclonal antibody against phosphoprotein of Borna disease virus[J].Chin J Biologicals,2013,(02):244-247.DOI:10.13200/j.cjb.2013.02.105.malh.020(in Chinese)马丽华,黄荣忠,张亮,等.抗博尔纳病病毒磷蛋白多克隆抗体的制备及鉴定[J].中国生物制品学杂志,2013,(02):244-247.DOI:10.13200/j.cjb.2013.02.105.malh.020