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

细胞系中组成型表达的核衣壳蛋白的分析

基本信息

批准号：
09670312
负责人：
NISHIO Machiko
金额：
$ 2.05万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670312/
关键词：
parainfluenza virus P protein NP protein L protein V protein monoclonal antibody RNA homotrimer homotrimer

项目摘要

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.

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。但是，尚未获得针对V或L蛋白的特异性mAB。因此，我制备了抗V或抗L蛋白mAb，并证明了V或L蛋白在病毒感染的细胞中的分布。2。通过抗P mAb和P蛋白缺失突变体的西部免疫印迹，确定了P-P相互作用所必需的区域。适当的折叠和同型聚合物需要围绕AA 211-248的P蛋白区域，该区域是由该区域中预测的盘绕螺旋介导的。3。通过使用抗NP MAB的西部免疫印迹，对NP蛋白的缺失突变体进行了分析，以识别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末端294 AA都是NP-NP自组装所必需的，并且P蛋白和另一个区域的两个C末端部分。 AA403-494，到P蛋白的N末端部分4。通过使用抗L MABS进行西部免疫印迹，我证明L蛋白直接与P和NP蛋白结合。此外，确定L蛋白结合在P或NP蛋白上的必需区域。与L蛋白结合需要P蛋白上的AA 278-353区。 NP蛋白上的AA 403-494区域与L蛋白结合需要。5。通过西北印迹分析，我证明了与RNA结合的P，V或NP蛋白上的区域。