呼吸道合胞病毒NS2蛋白单克隆抗体的制备及初步研究

Preparation and Preliminary Study of Monoclonal Antibodies Against Respiratory Syncytial Virus NS2

呼吸道合胞病毒(Respiratory syncytial virus,RSV)是一种引起婴幼儿下呼吸道感染的RNA病毒,其非结构蛋白(Nonstructural protein 2,NS2)在病毒复制和宿主抗病毒免疫中起到关键作用,但目前尚无商品化NS2单克隆抗体。本研究选取NS2基因,经密码子优化后构建重组质粒pET-32a-NS2。将该重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,经16℃诱导表达后,对表达蛋白进行SDS-PAGE鉴定。应用亲和层析技术纯化重组蛋白,将纯化的蛋白免疫小鼠制备单克隆抗体,并将该单克隆抗体用于Western blot分析、免疫荧光检测和免疫沉淀反应。结果表明,诱导后的重组蛋白为可溶性表达,并经纯化后得到高纯度的NS2蛋白;纯化的NS2蛋白免疫小鼠后,获得了一株特异性强、反应性好的RSV NS2单克隆抗体4A4 2B2,其亚型为IgG1,且抗体效价可达1∶1024000;使用该单克隆抗体鉴定出RSV NS2蛋白在转染和感染细胞中正常表达,且NS2主要分布在细胞质中,在细胞核周围形成点状聚集体;在免疫沉淀反应中,该单克隆抗体作为捕获抗体能够特异性结合细胞中表达的NS2蛋白,为NS2蛋白的功能研究奠定了基础。

The respiratory syncytial virus(RSV)is an RNA virus that causes infections in the lower respiratory tract in infants and young children.Nonstructural protein NS2 plays a crucial role in virus replication and host antiviral immunity,but a commercial NS2 monoclonal antibody is not available.We selected NS2 and constructed a recombinant plasmid(pET-32a-NS2)after codon optimization.The recombinant plasmid was transformed into Escherichia coli BL21(DE3)and we induced expression at 16℃.Expressed protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.We purified the recombinant protein using affinity chromatography,immunized mice with the purified protein to prepare monoclonal antibodies,and used the monoclonal antibodies for Western blot,immunofluorescence detection,and immunoprecipitation reactions.The induced recombinant protein was soluble and purified to obtain high-purity NS2 protein.After immunizing mice with the purified NS2 protein,a highly specific and reactive RSV NS2 monoclonal antibody(4A42B2)was screened,with a subtype of IgG1 and antibody titer of 1:1024000.We identified that RSV NS2 protein was expressed in transfected and infected cells using the prepared monoclonal antibody.NS2 protein was distributed mainly in the cytoplasm and formed puncta aggregates around nuclei.This monoclonal antibody could be applied as a capture antibody in immunoprecipitation reactions to bind specifically to Ns2 proteins expressed in cells.Our data lay a foundation for functional study of NS2 protein.