Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generat...Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.展开更多
基金supported by the Natural Science Foundation of Heilongjiang Province,China(JQ2019C005)the National Natural Science Foundation of China(31702265 and 32172847)。
文摘Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.
