抗人hnRNP A1单克隆抗体的制备及在颅咽管瘤组织中的应用

Preparation and Preliminary Application of Monoclonal Antibodies against Human Heterogeneous Nuclear Ribonucleoprotein A1

目的制备核不均一核糖核蛋白A1(heterogeneous nuclear ribonucleoprotein A1,hnRNP A1)的单克隆抗体,探讨hnRNP A1蛋白的生物学功能。方法利用重组hnRNP A1蛋白为免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2/0进行常规融合,通过间接ELISA的筛选和有限稀释克隆化,获得鼠抗hnRNPA1单克隆抗体的杂交瘤细胞株,通过ELISA、Western Blot和免疫组织化学实验等方法分别对其效价和特异性进行鉴定。结果成功地建立了3株稳定分泌抗hnRNPA1的单克隆抗体杂交瘤细胞株,分别命名为E006、E009和E012。3株单克隆抗体的免疫球蛋白亚类均为IgG1。3株单克隆抗体通过组织化学实验和Western Blot实验都能特异性地结合真核细胞内源性的hnRNPA1蛋白。结论获得了效价高、特异性好的抗hnRNP A1蛋白的单克隆抗体,这为进一步研究hnRNP A1的生物学功能及它在癌发生事件中的作用奠定了基础。

Objective To obtain monoclonal antibodies against heterogeneous nuclear ribonucleoprotein Al （hnRNPAl）for further study of the biological function of hnRNP Al protein, Methods BALB/c mice were immunized with recombinant hnRNPAl. The hybridoma cell lines secreting monoclonal antibodies against hnRNPAl were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting, and immunohistochemistry. Results Three hybridoma cell lines （E006, E009 and E012） stable in secreting specific monoclonal antibodies were successfully obtained. These three monoclonal antibodies are IgG1. They could specifically bind to endogenous hnRNPAl proteins proved by Western blotting and immunohistochemisty. Conclusion Monoclonal antibodies against hnRNPAl with high titers and specificity have been successfully prepared, which has laid the foundation for further study of hnRNPAl protein.