摘要
目的制备抗Ras相关蛋白RalB的单克隆抗体(McAb),探讨RalB蛋白在哺乳动物真核细胞中的表达,为进一步研究RalB功能奠定基础。方法以纯化的原核表达RalB蛋白免疫的BALB/c小鼠脾细胞与Sp2/0融合,通过间接ELISA的筛选和有限稀释克隆化,获得稳定分泌抗RalB蛋白的McAb的杂交瘤细胞株。通过免疫组织化学方法和Western-blot方法分别对RalB在组织中的表达及特异性进行鉴定。结果获得2株能高效分泌特异性McAb的抗RalB的杂交瘤细胞系F001,F002,其免疫球蛋白亚类均为IgG1。免疫组织化学实验结果表明RalB定位于肝癌细胞的细胞膜,Western-blot显示RalB在不同的肝癌细胞及正常肝细胞中均有表达。结论成功获得抗RalB的特异性的单克隆抗体,为进一步研究RalB与肿瘤的相关功能研究创造了条件。
Objective In order to explore the expression of RalB（ras related;GTP binding protein B）in mammal eucaryotic cell,we prepared and characterized monoclonal antibodies against RalB.Methods Hybridomas were generated by the fusion with Sp2/0 myelomas and spleen cells,which were from mice immunized with RalB recombinant proteins.The monoclonal antibodies against RalB were then used to identify the expression of RalB in mammal eucaryotic cell,including normal hepatic cell and hepatoma carcinoma cells,by Western blot and Immunohistochemistry.Results Two hybridoma cell lines,F001,F002,had been produced,each of which stably secrets antibodies against RalB.Subclass of IgG are both belonged to IgG1.Immunohistochemistry demonstrated that RalB was presented in plasma membrane of hepatoma tissue.Western-blot showed that RalB was expressed in all concerned cell.Conclusion The monoclonal antibodies against RalB protein have been successfully prepared,which should provide useful reagent for further investigation into the biological function of RalB.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2008年第4期651-653,670,共4页
Journal of Sichuan University(Medical Sciences)
关键词
RalB蛋白
单克隆抗体
真核细胞
表达
RalB Monoclonal antibody Mammal eucaryotic cell Presentation
