Analysis of the nucleocapsid proteins that are constitutively expressed in cell lines

1. 我已经分离了很多针对hPIV-2的P或NP蛋白的单克隆抗体。然而，目前还没有获得针对V或L蛋白的单克隆抗体。因此，我制备了抗V或抗L蛋白单克隆抗体，并证明了V或L蛋白在病毒感染细胞中的分布。通过使用抗P单克隆抗体和P蛋白缺失突变体进行Western免疫印迹，确定了P-P相互作用必需的区域。包含a211 -248的P蛋白区域是正确折叠和同源三聚体所必需的，这是由该区域预测的卷曲线圈介导的。通过使用抗NP单克隆抗体的Western免疫印迹技术，对NP蛋白的缺失突变体进行分析，以确定NP-NP相互作用所必需的区域和NP蛋白上的P蛋白结合位点。结果表明，NP蛋白的n端294aa都是NP- NP自组装所必需的，P蛋白的两个c端部分和另一个区域是NP- NP自组装所必需的。aa403-494，到P蛋白的n端部分。通过使用抗L单克隆抗体的Western免疫印迹，我证明了L蛋白直接与P和NP蛋白结合。此外，还确定了L蛋白与P或NP蛋白结合的必需区域。P蛋白上的a278 -353区域是与L蛋白结合所必需的。NP蛋白上的aa 403-494区域是与L蛋白结合所必需的。通过西北印迹法，我展示了P、V或NP蛋白上与RNA结合的区域。

1. I have already isolated a lot of monoclonal antibodies directed against the P or NP protein of hPIV-2. However, no MAbs specific for the V or L protein has been obtained. Therefore, I prepared anti-V or anti-L protein MAbs, and demonstrated the distribution of the V or L protein in virus-infected cells.2. By Western immunoblotting with anti-P MAbs and P protein deletion mutants, the region essential for P-P interactions was determined. The P protein region encompassing aa 211-248 was required for proper folding and homotrimer which is mediated by predicted coiled-coils in this region.3. By Western immunoblotting using anti-NP MAbs, the analysis of deletion mutants of the NP protein was performed to identify the region essential for NP-NP interaction and P protein-binding sites on the NP protein. The results indicate that the N-terminal 294 aa of the NP protein are all required for NP-Np self-assembly, and that two C-terminal parts of the P protein and another region. aa403-494, to the N-terminal part of the P protein.4. By Western immunoblotting using anti-L MAbs, I demonstrated that the L protein directly binds to the P and NP proteins. Furthermore, the essential region for the L protein binding on the P or NP protein was determined. The region aa 278-353 on the P protein is required for binding to the L protein. And the region aa 403-494 on the NP protein is required for binding to the L protein.5. By Northwestern blot assay, I demonstrated the regions on the P, V or NP protein which binds to RNA.