抗土拨鼠肝炎病毒核心蛋白单克隆抗体的制备、性质鉴定及初步应用

目的研制出能特异与土拨鼠肝炎病毒核心蛋白（WHc）结合的单克隆抗体,使之能特异性地对土拨鼠肝炎病毒（WHV）进行检测,并可能应用于相关肝炎病毒的筛查。方法以原核表达的土拨鼠肝炎病毒重组核心蛋白（WHc 1～149氨基酸）免疫BALB/c小鼠,常规杂交瘤技术进行细胞融合,有限稀释法克隆化,间接酶联免疫吸附试验（ELISA）和免疫组织化学（IHC）筛选、鉴定。结果筛选出5株（4B1E、6C5D、6C5C、6D1D、6D1G）能稳定分泌抗土拨鼠肝炎病毒核心蛋白抗体的杂交瘤细胞株。此5株单抗适用于ELISA、IHC、Western blot等方面的研究,与HBcAg有交叉反应,并且在部分中国旱獭的肝脏组织进行IHC检测呈现阳性反应。结论制备的5株单抗可用于土拨鼠肝炎病毒等嗜肝脱氧核糖核酸病毒的研究,可能在寻找新的相关肝炎病毒中起重要作用。

Development, characterization and application of monoclonal antibodies against woodchuck hepatitis virus core protein

Objective:To develop monoclonal antibodies against woodchuck hepatitis virus core protein which can be used to detect woodchuck hepatitis virus, and to carry out research on woodchuck hepatitis virus and related hepatitis virus.Methods:BALB/c mice were immunized with prokaryotically expressed recombinant woodchuck hepatitis virus core protein(WHc 1-149), mouse spleen cells were fused with SP2/0 cells by a standard hybridoma technique. Positive hybridomas that produced antobodies against woodchuck hepatitis virus core protein were screened by ELISA and immunohistochemistry. Cloning procedure was carried out using the method of limiting dilution.Results:5 hybridoma clones stably producing monoclonal antobodies(4B1E,6C5D,6C5C,6D1D,6D1G) against woodchuck hepatitis virus core protein were established. All these antibodies were shown to be good for ELISA, IHC, and Western blot assays and found cross-reactive between HBcAg and 5 monoclonal antobodies by our experiments. Using these antibodies we were able to detect positive signal in liver tissue of some Chinese Marmots by IHC.Conclusion:All these 5 monoclonal antibodies we developed can be used for WHV or Hepadnavirus study, and may also serve as an important tool for discovering new relative hepadnaviruses.