西尼罗河病毒preM/EⅢ融合蛋白特异性单克隆抗体的制备以及应用分析

目的制备针对西尼罗河病毒preM/EⅢ融合蛋白特异性单克隆抗体,并分析其在临床检测中的应用价值。方法采用原核表达系统表达西尼罗河病毒preM/E融合蛋白,作为抗原,免疫BALB/c小鼠,按常规单克隆抗体技术将免疫后小鼠脾细胞与SP2/0细胞融合,采用有限稀释法筛选能稳定分泌抗preM/EⅢ单抗的杂交瘤细胞株。接种杂交瘤细胞于小鼠腹腔,抽取腹水纯化单克隆抗体。采用间接ELISA和Western blotting对抗体特异性和效价进行分析,并对临床样本进行检测。结果获得了1株能稳定分泌preM/EⅢ蛋白单克隆抗体的杂交瘤细胞株,命名为ME1。纯化后抗体效价为10-6,其亚类为IgG1。ME1单抗能与西尼罗病毒preM/EⅢ蛋白以及西尼罗病毒发生特异性反应,而与日本脑炎病毒(JEV)、圣路易斯脑炎病毒(SLEV)、黄热病毒(YFV)和登革热病毒(DENV)无交叉反应。临床诊断准确率为97.78%。结论 ME1单克隆抗体具有较高的特异性,临床诊断准确性较高,具有临床应用价值。

Preparation of a monoclonal antibody against fusion protein preM/EIII of WNV

Objective To prepare a monoclonal antibody(mAb) against the fusion protein preM/EⅢ of West Nile virus(WNV) for clinical detection of WNV.Methods Sp2/0 cells were fused with the spleen cells of BALB/c mice immunized with the recombinant fusion protein preM/EIII expressed in E.coil to obtain the hybridoma cell line that secreted preM/EⅢ mAb.The hybridoma cells were injected into the peritoneal cavity of BALB/c mice and the ascites was collected and purified.The specificity and titer of the obtained mAb were determined using ELISA and Western blotting.Results One hybridoma cell line secreting preM/EIII mAb,named ME1,was obtained.The titer of the purified mAb was 10-6.Identified as a mAb of the Ig subclass IgG1,ME1 was capable of specific reactions with preM/EIII protein and WNV without cross-reactions with other viruses such as JEV,SLEV,YFV and DENV.The accuracy of clinical testing of MNV with ME1 was 97.78%.Conclusion The mAb against preM/EⅢ obtained have a high specificity and accuracy in clinical testing of MNV and can be used in clinical diagnosis of MNV infection.