Chen Zheng, Shao Lin, Ye Jing, Li Yongmao, Huang Shaomei, Chen Huanchun, Cao Shengbo
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, China.
Hybridoma (Larchmt). 2012 Apr;31(2):137-41. doi: 10.1089/hyb.2011.0107.
Non-structural proteins NS3 and NS5 of Japanese encephalitis virus (JEV) were expressed in Escherichia coli and purified by dialysis. Two monoclonal antibodies (MAbs) named 1H7 and 2D4 against NS3 protein and three MAbs named 3C4, 3H7, and 3F10 against NS5 protein were generated by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from NS3 or NS5 protein immunized mice. Then activity of MAbs was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and indirect immunofluorescent assays (IFA). Our results demonstrated that all the MAbs showed high specificity and sensitivity in IFA at 1:100 dilution and in Western blot analysis at 1:500 dilution, which indicated that these MAbs against NS3 and NS5 proteins of JEV may be used as valuable tools for analysis of the protein functions and pathogenesis of JEV.
日本脑炎病毒（JEV）的非结构蛋白NS3和NS5在大肠杆菌中表达，并通过透析进行纯化。通过将小鼠骨髓瘤细胞系SP2/0与经NS3或NS5蛋白免疫的小鼠的脾淋巴细胞融合，产生了两种针对NS3蛋白的单克隆抗体（MAb），分别命名为1H7和2D4，以及三种针对NS5蛋白的单克隆抗体，分别命名为3C4、3H7和3F10。然后通过酶联免疫吸附测定（ELISA）、蛋白质印迹分析和间接免疫荧光测定（IFA）对单克隆抗体的活性进行了表征。我们的结果表明，所有单克隆抗体在1:100稀释的IFA和1:500稀释的蛋白质印迹分析中均显示出高特异性和敏感性，这表明这些针对JEV的NS3和NS5蛋白的单克隆抗体可作为分析JEV蛋白功能和发病机制的有价值工具。
