口蹄疫病毒非结构蛋白3A单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against nonstructural protein 3A of foot-and-mouth disease virus

以原核表达并纯化的口蹄疫病毒(FMDV)非结构蛋白3A免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用间接ELISA方法筛选杂交瘤细胞,有限稀释法进行亚克隆,获得3株能稳定分泌抗3A蛋白单抗的杂交瘤细胞,分别命名为3H2、4B1、7F5,并对这3株单抗进行了生物学鉴定。结果显示,单抗3H2和4B1为IgG2a亚型,7F5为IgG2b亚型。单抗7F5针对的表位与3H2和4B1两株单抗的表位不一致;这3株单抗均能够与FMDV特异性结合,且单抗4B1与所有3A相关蛋白均反应。稳定性鉴定结果表明,获得的3株杂交瘤细胞均能稳定分泌单克隆抗体。研究结果为进一步探索3A蛋白的结构和功能以及建立诊断方法奠定了基础。

BALB/c mice were immunized with foot-and-mouth disease virus（FMDV） nonstructural protein 3A expressed in Escherichia coli.Splenocytes of the BALB/c mice were fused with myeloma cells SP2/0 for producing hybridoma.Specific antibody was determined by indirect ELISA and positive hybridoma cells were cloned by limiting dilution assay.Three hybridoma cell lines stably secreting monoclonal antibodies（McAbs） against the protein 3A were developed and named 3H2,4B1 and 7F5,respectively.The McAb 7F5 was classified into subtype IgG2b,while 3H2 and 4B1 were identified to be subtype IgG2a.7F5 could recognize different epitope from the other strains.All the McAbs could react with FMDV in IFA.The result of indirect ELISA and Western-blot showed that these McAbs bound specifically to the protein 3A.It was concluded from the above-mentioned result that the prepared McAbs against the protein 3A could not only provide a foundation for studying the structure and function of FMDV,but also a mean for development of diagnostic methods.