禽流感病毒特异性NP单克隆抗体的鉴定及禽流感特异性检测方法的建立
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摘要
目的筛选鉴定禽流感病毒特异性单克隆抗体(单抗)并建立一种适合禽源流感病毒特异性检测的抗原检测方法。方法利用分子进化树分析方法对甲型流感病毒核蛋白(NP)基因进行分类,从禽流感病毒分支和人流感病毒分支上各挑选一个代表性流感病毒株的NP基因进行重组抗原的表达及其单抗的筛选制备,最后应用免疫渗滤技术(A-Dot-ELISA)建立抗原快速检测方法。结果根据分子进化树分析结果确定禽流感H5N1病毒株HK/212/2003和人流感H1N1病毒株CA/04/2009的NP基因进行原核表达,获得高纯度的禽流感病毒NP重组抗原rNP-HK/212和人流感病毒NP重组抗原rNP-CA/04;利用上述两种NP抗原制备出抗rNP-HK/212单抗53株,通过差异筛选获得只对禽流感病毒NP蛋白rNP-HK/212有特异性反应而不与人流感病毒NP反应的3株单抗(1F2,5D2及25A2);免疫荧光方法证实1F2等3株单抗只特异识别禽流感病毒;利用单抗1F2成功建立一种适于禽流感病毒特异性检测的抗原快速检测方法 AIV-Dot-ELISA,该方法对病毒滴度为0.025~0.1HA titer的19个不同亚型的禽流感病毒均能检出,对病毒滴度为5HA titer的8个人流感病毒和2个乙型流感病毒的检测均为阴性。结论本研究成功制备出禽流感病毒NP蛋白特异性单抗,并建立了一种仅特异识别禽流感病毒的抗原快速检测方法 AIV-Dot-ELISA,为快速鉴别禽类流感病毒和人类流感病毒感染提供了一种有用的分析检测工具。
Avian influenza A virus strain HK/212/2003(H5N1)and human influenza A virus strain California/04/2009(H1N1)were chosen for expression of recombinant NP antigen and preparation of anti-NP monoclonal antibodies(mAbs).Fifty-three anti-NP mAbs were obtained by immunizing BALB/c mouse with recombinant NP antigen of rNP-HK/212.All mAbs were characterized with avian influenza A virus recombinant NP antigen rNP-HK/212 and human influenza A virus recombinant NP antigen rNP-CA/04 in ELISA and divided further into three classes based on their reactivity to the two recombinant NP antigens.Forty-five of the ClassⅠ mAbs reacted both with rNP-HK/212 and rNP-CA/04.Three of the ClassⅡ mAbs reacted only with rNP-HK/212 but not rNP-CA/04,implying that ClassⅡ mAbs might recognize a specific epitope on NP of avian influenza A virus.Five of the ClassⅢ mAbs reacted with both two recombinant NP antigens but had weaker reactivity with rNPHK/212.Then,a rapid test for detection of avian influenza A virus was developed based on an enzyme immune-filtration system,AIV-Dot-ELISA.This test was demonstrated to have reactivity with all nineteen different subtypes of influenza A virus strains at the virus titer of 0.025 to 0.1HA titer,but negative reactivity with eight human influenza A virus strains and two influenza B virus strains even at the virus titer of 5HA titer.Our results will provide a tool to directly differentiate infection by avian influenza A virus from human influenza virus.
Avian influenza A virus strain HK/212/2003(H5N1)and human influenza A virus strain California/04/2009(H1N1)were chosen for expression of recombinant NP antigen and preparation of anti-NP monoclonal antibodies(mAbs).Fifty-three anti-NP mAbs were obtained by immunizing BALB/c mouse with recombinant NP antigen of rNP-HK/212.All mAbs were characterized with avian influenza A virus recombinant NP antigen rNP-HK/212 and human influenza A virus recombinant NP antigen rNP-CA/04 in ELISA and divided further into three classes based on their reactivity to the two recombinant NP antigens.Forty-five of the ClassⅠ mAbs reacted both with rNP-HK/212 and rNP-CA/04.Three of the ClassⅡ mAbs reacted only with rNP-HK/212 but not rNP-CA/04,implying that ClassⅡ mAbs might recognize a specific epitope on NP of avian influenza A virus.Five of the ClassⅢ mAbs reacted with both two recombinant NP antigens but had weaker reactivity with rNPHK/212.Then,a rapid test for detection of avian influenza A virus was developed based on an enzyme immune-filtration system,AIV-Dot-ELISA.This test was demonstrated to have reactivity with all nineteen different subtypes of influenza A virus strains at the virus titer of 0.025 to 0.1HA titer,but negative reactivity with eight human influenza A virus strains and two influenza B virus strains even at the virus titer of 5HA titer.Our results will provide a tool to directly differentiate infection by avian influenza A virus from human influenza virus.
引文
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[3]Fraser C,Donnelly CA,Cauchemez S,et al.Pandemic potential of a strain of influenza A(H1N1):early findings[J].Science,2009,324(5934):1557-1561.DOI:10.1126/science.1176062
[4]Muscatello DJ,Cretikos MA,Macintyre CR.All-cause mortality during first wave of pandemic(H1N1)2009,New South Wales,Australia,2009[J].Emerg Infect Dis,2010,16(9):1396-1402.DOI:10.3201/eid1609.091723
[5]Alexander DJ.A review of avian influenza in different bird species[J].Vet Microbiol,2000,74(1-2):3-13.DOI:10.1016/S0378-1135(00)00160-7
[6]Li KS,Guan Y,Wang J,et al.Genesis of a highly pathogenic and potentially pandemic H5N1influenza virus in eastern Asia[J].Nature,2004,430(6996):209-213.DOI:10.1038/nature02746
[7]Gao R,Cao B,Hu Y,et al.Human infection with a novel avianorigin influenza A(H7N9)virus[J].N Engl J Med,2013,368(20):1888-1897.DOI:10.1056/NEJMoa1304459
[8]Butt KM,Smith GJ,Chen H,et al.Human infection with an avian H9N2influenza A virus in Hong Kong in 2003[J].J Clin Microbiol,2005,43(11):5760-5767.DOI:10.1128/JCM.43.11.5760-5767.2005
[9]Du Ry van Beest Holle M,Meijer A,Koopmans M,et al.Human-to-human transmission of avian influenza A/H7N7,The Netherlands,2003[J].Euro Surveill,2005,10(12):264-268.
[10]Parry J.H10N8avian flu virus claims its first known human casualty[J].BMJ,2014,348:g1360.DOI:10.1136/bmj.g1360
[11]Reina J,Padilla E,Alonso F,et al.Evaluation of a new dot blot enzyme immunoassay(directigen flu A+B)for simultaneous and differential detection of influenza a and B virus antigens from respiratory samples[J].J Clin Microbiol,2002,40(9):3515-35157.DOI:10.1128/JCM.40.9.3515-3517.2002
[12]Chen Y,Xu F,Fan X,et al.Evaluation of a rapid test for detection of H5N1avian influenza virus[J].J Virol Methods,2008,154(1-2):213-215.DOI:10.1016/j.jviromet.2008.08.013
[13]Chen Y,Xu F,Gui X,et al.A rapid test for the detection of influenza A virus including pandemic influenza A/H1N1 2009[J].J Virol Methods,2010,167(1):100-102.DOI:10.1016/j.jviromet.2010.02.001
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