猫杯状病毒VP1蛋白单克隆抗体的制备及其抗原表位的鉴定

发布时间：2018-04-08 22:32

本文选题：猫杯状病毒　切入点：VP-E蛋白　出处：《中国预防兽医学报》2017年12期

【摘要】：为制备抗猫杯状病毒(FCV)衣壳蛋白VP1的单克隆抗体(MAb)及鉴定其表位,本研究以原核表达的重组VP1-E蛋白(aa427~aa524)免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞(SP2/0)进行融合制备杂交瘤细胞;以纯化的FCV-2280病毒粒子作为检测抗原,采用间接ELISA检测方法筛选杂交瘤细胞株,分析杂交瘤细胞所分泌的抗体亚型、中和活性及所识别的抗原表位等。结果表明,获得两株稳定分泌抗FCV VP1-E MAbs的杂交瘤细胞E2F1和E2F6,抗体均为IgG1亚型,轻链为κ链。杂交瘤细胞分泌的抗体无中和活性。Western blot结果显示,两种抗体可以特异性识别FCV-2280,表明其针对的抗原表位为线性表位。两株MAb识别VP1-E序列均为~(467)YICGSLQRAWG~(477)。生物信息学分析显示该抗原表位在FCV VP1蛋白中相对保守。该MAb为建立特异性强、灵敏度高的FCV的检测方法奠定基础。
[Abstract]:In order to prepare monoclonal antibody against capsid protein VP1 and identify its epitopes, BALB/c mice were immunized with prokaryotic expressed recombinant VP1-E protein (aa427a524). The spleen cells were fused with myeloma cells (SP2 / 0) to produce hybridoma cells.Using purified FCV-2280 virus particles as detection antigen, indirect ELISA assay was used to screen hybridoma cell lines, and antibody subtypes, neutralizing activities and antigen epitopes recognized by hybridoma cells were analyzed.The results showed that two hybridoma cells, E2F1 and E2F6 secreted stably against FCV VP1-E MAbs, were obtained. The antibody was IgG1 subtype and the light chain was 魏 chain.The antibody secreted by hybridoma cells had no neutralization activity. Western blot results showed that the two antibodies could specifically recognize FCV-2280, indicating that the antigenic epitopes were linear epitopes.The VP1-E sequences of the two strains identified by MAb were both 467 (YICGSLQRAWGN).Bioinformatics analysis showed that the epitope was relatively conserved in FCV VP1 protein.The MAb lays a foundation for the establishment of a specific and sensitive method for the detection of FCV.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室;
【基金】：国家重点研发项目(2016YFD0501001) 中央级公益性科研院所基本科研业务费专项(1610302017012)
【分类号】：S852.65

【相似文献】