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。 Objective To screen and identify specific monoclonal antibodies against avian influenza virus (McAb) and to establish an antigen detection method suitable for the specific detection of avian influenza virus. Methods The nucleoprotein (NP) gene of influenza A virus was classified by molecular phylogenetic tree analysis. NP gene of each representative influenza virus strain was selected from the branch of avian influenza virus and the branch of human influenza virus for recombinant antigen expression Monoclonal antibody screening and preparation, and finally using immunodialysis (A-Dot-ELISA) to establish a rapid antigen detection method. Results The NP gene of avian influenza virus H5N1 strain HK / 212/2003 and human influenza H1N1 strain CA / 04/2009 was confirmed by molecular phylogenetic tree analysis. The NP gene of avian influenza virus NP recombinant antigen rNP-HK / 212 and human recombinant influenza virus NP recombinant antigen rNP-CA / 04; using the above two NP antigen prepared 53 strains of anti-rNP-HK / 212 monoclonal antibody, obtained by differential screening only against the bird flu virus NP protein rNP-HK / 212 Three monoclonal antibodies (1F2, 5D2 and 25A2) that reacted specifically but not with human influenza virus (NP) were confirmed by immunofluorescence assay. Three monoclonal antibodies (McAb), such as 1F2, were found to specifically recognize the avian influenza virus AIV-Dot-ELISA, a rapid antigen detection method for avian influenza virus-specific detection, was able to detect 19 different subtypes of avian influenza viruses with a titer of 0.025-0.1HA titer. The virus titer was The 5HA titer of 8 human influenza viruses and 2 influenza B viruses were negative. Conclusions This study successfully prepared the NP protein-specific monoclonal antibody of avian influenza virus and established a rapid detection method AIV-Dot-ELISA for antigen-specific detection of avian influenza virus for the rapid identification of avian influenza virus and human influenza virus infection A useful analysis of testing tools.