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Influenza virus is a major threat to human health worldwide.The nucleocapsid protein(NP) of influenza A(Flu A) virus plays an essential role in the replication of its viral RNA.Due to its high degree of ho mology,NP protein is used as a key target antigen to develop rapid tests for the viral infection.The B-cell antigenic epitopes of the NP protein are,however,still poorly understood due to its complex conformation.In this study,a recombinant NP antigen derived from the H1N1 influenza strain A/Califoruia/04/2009 was expressed and used to generate a panel of monoclonal antibodies(mAbs) against NP protein of Flu A virus.One specific mAb,designated as 4D2,was identified by epitope mapping with a series of overlapping synthetic peptides.Reactivity towards synthetic peptides indicated that the epitope for 4D2 is linear.Nine continuous amino acids recognized by 4D2 were finally located in the C terminal of NP.The NP amino acid sequence alignment of 3 144 Flu A viruses,from 2005.9 to 2009.5 in GenBank demonstrated, that this linear epitope is highly conserved among different strains of Flu A virus.Immunofluorescence and immunohistochemical experiments also indicated 4D2 is strong specific to NP antigen in living cells of fixed tissues when infected by Flu A virus.Taken together, these results demonstrated the usefulness of 4D2 in clinical diagnostic applications and as a tool for further study of the structure and function of Flu A virus.This is the first report that a linear epitope exists on NP protein of Flu A virus.Much work is needed to understand the function and importance of this epitope with respect to the virus replication and transcription