呼吸道合胞病毒NS1蛋白鼠单克隆抗体的制备

Preparation of a mouse monoclonal antibody against the NS1 protein of respiratory syncytial virus

摘要:

本研究旨在制备抗呼吸道合胞病毒(respiratory syncytial virus, RSV)非结构蛋白1 (nonstructural protein 1, NS1)单克隆抗体，以分析在转染和感染过程中NS1蛋白的表达和分布情况，并评估其在免疫沉淀反应中的应用价值。将NS1基因片段构建至原核表达质粒，经大肠杆菌表达、亲和层析纯化后得到NS1蛋白，将纯化的蛋白免疫小鼠后，通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)技术筛选能稳定分泌NS1单克隆抗体的杂交瘤细胞株，并将该单抗用于间接免疫荧光(indirect immunofluorescence assay, IFA)与免疫印迹(Western blotting)检测，分析RSV NS1在过表达细胞和病毒感染细胞中的表达与分布情况，评估该单抗在免疫沉淀反应中的应用价值。结果发现，RSV NS1蛋白在大肠杆菌中成功表达及纯化，免疫小鼠后，获得了一株特异性强、反应性好的RSV NS1单抗，其亚型为IgG1，且抗体效价可达1:15 360 000；使用该单抗鉴定出RSV NS1蛋白在转染和感染细胞中正常表达，IFA结果表明，NS1主要分布在细胞质与细胞核；并验证该单抗在免疫沉淀反应中作为捕获抗体能够特异性结合细胞中表达的NS1蛋白。本研究经原核表达系统成功获得RSV NS1蛋白，成功制备出抗RSV NS1单抗，该抗体能特异性识别NS1蛋白，可被应用于免疫沉淀方法，为NS1蛋白的功能研究奠定了基础。

Abstract:

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody’s application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.