National Institute of Diagnostics and Vaccine Development in Infectious Disease, State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, China.
J Med Virol. 2014 Jun;86(6):995-1002. doi: 10.1002/jmv.23812. Epub 2013 Oct 17.
Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope.
流感病毒仍然对全球人类健康构成重大威胁。甲型流感病毒的核蛋白（NP）在病毒复制和转录中发挥着重要作用，因此成为有前途的治疗靶点。NP 在天然条件下形成复杂构象，在研究 NP 功能的免疫印迹等免疫测定中可能变性。因此，制作识别线性表位而不是构象表位的 NP 特异性单克隆抗体（mAb）是很有用的。在这项研究中，甲型流感病毒的重组 NP（rNP）被过表达，并用于产生一组抗 NP mAb。这些抗 NP mAb 根据其在 Western blot 中的反应性分为三类。只有一类 mAb 可与线性 rNP 片段反应。一类 mAb 中的一种，4D2，通过一系列重叠合成肽的表位作图进一步表征，表明 4D2 表位是位于氨基酸残基 243 和 251 之间的暴露于表面的线性表位。该表位在不同的甲型流感病毒中高度保守，同一性为 98.4%（17,922/18,210）。Western blot、共免疫沉淀、免疫荧光和免疫组织化学实验均表明 4D2 高度特异性地针对甲型流感病毒的 NP。结果表明，4D2 可作为研究工具，用于研究甲型流感病毒复制周期中 NP 的功能。需要进一步的工作来了解该表位的功能和重要性。
