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

1.我已经分离了许多针对hPIV-2的P或NP蛋白的单克隆抗体。然而，尚未获得特异于V或L蛋白的MAbs。因此，本研究制备了抗V或抗L蛋白的单克隆抗体，并证明了V或L蛋白在病毒感染细胞中的分布.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相互作用所必需的区域。P蛋白211-248位氨基酸区域是正确折叠和同源三聚体所必需的，而同源三聚体是由该区域的卷曲螺旋介导的.通过使用抗NP单克隆抗体的Western免疫印迹，对NP蛋白的缺失突变体进行分析，以鉴定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N端294个氨基酸都是NP-Np自组装所必需的，而P蛋白C端的两个氨基酸和另一个氨基酸是NP-Np自组装所必需的。aa 403 -494，连接到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蛋白直接结合到P和NP蛋白。此外，确定了L蛋白结合P或NP蛋白的必需区域。P蛋白上的aa 278-353区域是与L蛋白结合所必需的。NP蛋白上的氨基酸403-494区域是与L蛋白结合所必需的.通过Northwestern印迹分析，我证明了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.