西尼罗病毒NS1单克隆抗体的制备及鉴定

Preparation and specificity analysis of the monoclonal antibodies against nonstructural 1 protein of West Nile virus

目的以西尼罗病毒非结构蛋白(nonstructural protein1,NS1)为免疫原制备西尼罗病毒NS1蛋白特异性单克隆抗体,并鉴定其特异性。方法在表达具有良好抗原性的重组西尼罗病毒NS1蛋白基础上免疫Balb/c小鼠,取免疫小鼠的脾细胞与小鼠骨髓瘤细胞进行融合,用间接ELISA法筛选阳性的杂交瘤细胞,并结合免疫荧光(IFA)和免疫印迹对所获得单克隆抗体的特异性进行鉴定,通过竞争抑制试验对m Ab识别的抗原位点进行分析。结果获得39株特异性针对西尼罗病毒NS1蛋白的单克隆抗体,Ig亚类测定结果Ig G3和Ig G2a单抗各2株,Ig G2b单抗5株,Ig G单抗1株,另外29株均为Ig G1。结论成功获得了特异性针对西尼罗病毒NS1蛋白的单克隆抗体,将为进一步建立西尼罗病毒NS1抗原检测方法及探讨NS1蛋白及抗体在西尼罗病毒发病机制中提供依据。

The early diagnosis of West Nile virus （WNV） infection is important for successful clinical management and epidemiological control. The non-structural protein 1 （NS1） of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and acts as a useful early diagnostic marker. In this study, we aimed to prepare monoclonal antibodies （mAbs） against NS1 protein of WNV and identify its specificity, as well as corresponding epitope. Firstly, Balb/c mice were immunized with recombinant WNV-NS 1 protein. The splenocytes and myeloma cells from immunized mice were fused to generate hybridoma cell lines, and the specificity of mAbs against WNV-NS1 was identified by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence assay （IFA） and Western blot analysis （WB） were further applied to identify the specificity of mAbs. Thirty-nine mAbs recognizing thirteen different antigenic epitopes on WNV-NS1 were obtained. These mAbs displayed exclusive specificity to WNV-NS1 by showing no cross-reaction with dengue viruses （DENV）, yellow fever virus （YFV） or Japanese encephalitis virus （JEV）. Five mAbs were identified as IgG2b subtype, two as IgG2a, two as IgG3, one as IgG and other twenty-nine as IgG1. Taken together, a group of monoclonal antibodies against WNV-NSI are successfully established which provides a potential value for early diagnosis and vaccine research of West Nile virus infection.