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.
摘要：
本研究的目的是制备针对呼吸道合胞病毒（RSV）非结构蛋白1（NS1）的小鼠单克隆抗体，以分析其在转染和感染过程中的表达和分布。此外,我们旨在评估抗体在免疫沉淀测定中的应用。首先,将NS1基因片段克隆到原核质粒中，并在大肠杆菌中表达。然后通过亲和层析纯化所得的NS1蛋白,用于免疫BALB/c小鼠。随后，使用间接酶联免疫吸附测定（ELISA）选择能够稳定分泌NS1单克隆抗体的杂交瘤细胞。该单克隆抗体用于间接免疫荧光测定（IFA）和Western印迹，以分析RSVNS1在过表达和感染细胞中的表达和分布。最后,该单克隆抗体的可靠性通过免疫沉淀试验进行评估.结果表明，成功表达并纯化了RSVNS1蛋白。用这种蛋白质免疫小鼠后，我们获得了高度特异性的RSVNS1单克隆抗体，属于IgG1亚型，抗体滴度为1:15360000。使用这种单克隆抗体，在转染和感染的细胞中均鉴定出RSVNS1蛋白。IFA结果显示NS1在细胞质和细胞核中的主要分布。此外，我们证实了这种单克隆抗体可以有效地特异性结合细胞裂解物中的NS1蛋白，使其适合作为免疫沉淀测定中的捕获抗体。总之,我们的研究成功地通过原核表达系统实现了RSVNS1蛋白的生产，并制备了针对NS1的特异性单克隆抗体。该抗体证明了特异性鉴定NS1蛋白的能力，可用于免疫沉淀测定，从而为NS1蛋白的功能研究奠定基础。
