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： To identify the epitope on feline calicivirus（FCV-2280） VP1 protein, the monoclonal antibody （MAb） against the VP1 protein was prepared by fusion of SP2/0 cells with the splenic cells from immunized BALB/c mice with the truncated protein of VP1-E within aa427 to aa524 expressed in E.coli. Hybridoma cell lines stably secreting MAbs, named E2F1 and E2F6, were screened by indirect ELISA assay coated with the purified FCV-2280 virus particles. While the subtype, neutralizing activity of monoclonal antibody, the titer of the supernatant and ascites, and the epitope was analyzed. The result suggested the MAbs were IgG1 subtybe with K chain. Western blot assay showed that the MAbs were able to recognize FCV-2280 specifically, but had no virus neutralizing activity. Furthermore, the results displayed that the MAb recognized the linear epitope at 467yICGSLQRAWG477 in VP1-E. The epitope was relatively conserved among FCV strains. The preparation and identification of MAbs against the epitope of FCV in this study provided useful tools to detect FCV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2017年第12期1017-1021,共5页
Chinese Journal of Preventive Veterinary Medicine
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)
