西尼罗病毒E蛋白结构域Ⅲ特异性单克隆抗体的研制

目的研制针对西尼罗Ⅲ重组融合蛋白的特异性单克隆抗体。方法用西尼罗Ⅲ重组融合蛋白免疫BALB／c小鼠，用淋巴细胞杂交瘤技术进行细胞融合，并用间接ELISA方法进行筛选，所获得的单抗经间接ELISA、免疫印迹和间接免疫荧光试验进行鉴定。结果共获得了3株能稳定分泌针对西尼罗河病毒E蛋白结构域Ⅲ的特异性单抗的杂交瘤细胞株，分别命名为4F7、6H3和8FA，其单抗亚类鉴定分别属于IgG1、IgG1和Ig2a。这3株单克隆抗体均能与重组西尼罗河病毒E蛋白结构域Ⅲ发生特异性反应，而与日本脑炎病毒无交叉反应，并且，8FA和6H3识别同一抗原位点，4F7识别西尼罗河病毒E蛋白结构域Ⅲ的另一抗原位点。结论本实验成功研制出针对西尼罗河病毒E蛋白结构域Ⅲ的特异性单克隆抗体，为我国建立西尼罗河病毒的病原学检测方法并与日本脑炎病毒进行鉴别诊断提供了技术贮备。

Preparation of monoclonal antibodies against West Nile virus envelope protein domain Ⅲ

BACKGROUND: To prepare monoclonal antibodies against West Nile virus (WNV) envelope protein domain. METHODS: BALB/c mice were immunized with recombinant antigen of West Nile virus envelope protein domain, and the spleen cells of the mice were used to prepare the monoclonal antibodies (McAb) by hybridoma technique. RESULTS: Three hybridoma cell strains secreting McAbs against WNV envelope protein domain, designated as 4F7, 6H3 and 8E4, respectively, were obtained and were identified by indirect enzyme linked immunosorbent assay (ELISA), they belonged to IgG1, IgG1 and Ig2a, respectively. Two epitopes of envelope protein domain were determined, among them, 4F7 and 6H3 were against the same epitope and 8E4 to another one. CONCLUSIONS: The results of indirect ELISA, Western blot and indirect immunofluorescence experiment indicated that these three McAbs were specific for West Nile virus envelope protein domain and did not cross-react with Japanese encephalitis virus and other viruses, so they can be used for specific detection of West Nile virus.