CAJ | 학술논문

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0．025～0．1HAtiter的19个不同亚型的禽流感病毒均能检出，对病毒滴度为5HAtiter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。
목적：사선감정금류감병독특이성단극륭항체（단항）병건립일충괄합금원류감병독특이성검측적항원검측방법。방법이용분자진화수분석방법대갑형류감병독핵단백（NP）기인진행분류，종금류감병독분지화인류감병독분지상각도선일개대표성류감병독주적NP기인진행중조항원적표체급기단항적사선제비，최후응용면역삼려기술（A-Dot-ELISA）건립항원쾌속검측방법。결과근거분자진화수분석결과학정금류감H5N1병독주HK/212/2003화인류감H1N1병독주CA/04/2009적NP기인진행원핵표체，획득고순도적금류감병독NP중조항원rNP-HK/212화인류감병독NP중조항원rNP-CA/04；이용상술량충NP항원제비출항rNP-HK/212단항53주，통과차이사선획득지대금류감병독NP단백rNP-HK/212유특이성반응이불여인류감병독NP반응적3주단항（1F2，5D2급25A2）；면역형광방법증실1F2등3주단항지특이식별금류감병독；이용단항1F2성공건립일충괄우금류감병독특이성검측적항원쾌속검측방법AIV-Dot-ELISA，해방법대병독적도위0．025～0．1HAtiter적19개불동아형적금류감병독균능검출，대병독적도위5HAtiter적8개인류감병독화2개을형류감병독적검측균위음성。결론본연구성공제비출금류감병독NP단백특이성단항，병건립료일충부특이식별금류감병독적항원쾌속검측방법AIV-Dot-ELISA，위쾌속감별금류류감병독화인류류감병독감염제공료일충유용적분석검측공구。
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) .Fif-ty-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 an-tigens .Forty-five of the Class Ⅰ mAbs reacted both with rNP-HK/212 and rNP-CA/04 .Three of the Class Ⅱ mAbs reacted only with rNP-HK/212 but not rNP-CA/04 ,implying that Class Ⅱ mAbs might recognize a specific epitope on NP of avian in-fluenza A virus .Five of the Class Ⅲ mAbs reacted with both two recombinant NP antigens but had weaker reactivity with rNP-HK/212 .Then ,a rapid test for detection of avian influenza A virus was developed based on an enzyme immune-filtration sys-tem ,AIV-Dot-ELISA .This test was demonstrated to have reac-tivity with all nineteen different subtypes of influenza A virus strains at the virus titer of 0 .025 to 0 .1 HA titer ,but negative reactivity with eight human influenza A virus strains and two in-fluenza B virus strains even at the virus titer of 5 HA titer .Our results will provide a tool to directly differentiate infection by avian influenza A virus from human influenza virus .