小鼠抗人磷脂酰肌醇3激酶相互作用蛋白1单克隆抗体的制备和鉴定

Generation and characterization of monoclonal antibody against human PIK3IP1

目的:制备鼠抗人磷脂酰肌醇3激酶相互作用蛋白1(PIK3IP1)的单克隆抗体(mAb),探讨PIK3IP1蛋白的结构和功能。方法:利用重组蛋白GST-PIK3IP162-168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,获得鼠抗人PIK3IP1蛋白mAb的杂交瘤细胞株,通过ELISA,Westernblot和免疫荧光技术等方法对其特性进行鉴定。结果:成功地建立了1株稳定分泌抗PIK3IP1蛋白的mAb杂交瘤细胞株,命名为5C6,其免疫球蛋白亚类为IgG1。ELISA检测的腹水效价可达1:107。5C6mAb与重组GST-PIK3IP162-168蛋白有较强的特异性反应,而与大肠杆菌裂解液以及谷胱苷肽2S转移酶(GST)没有交叉反应。同时,5C6mAb也能特异性地结合真核细胞超表达的全长PIK3IP1蛋白和变异体PIK3IP1-v1,但检测不到内源性的PIK3IP1蛋白。共聚焦免疫荧光显微镜结果显示PIK3IP1和PIK3IP1-v1定位于胞质,在胞质中呈现不均一的斑点状分布。结论:获得了效价高、特异性好的PIK3IP1蛋白的mAb,为进一步研究PIK3IP1的生物学功能提供了有用的工具。

AIM：To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein.METHODS：BALB/c mice were immuonized with recombinant GST-PIK3IP162-168,Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach.The specificities of the monoclonal antibody was determined by ELISA,Western blot and Immunofluorescecence assay.RESULTS：One hybridoma cell line （5C6） stable in secreting specific monoclonal antibody was successfully obtained.The subclass of IgG belonged to IgG1.The ascite titers of this monoclonal antibody reached 1：107.It could specifically bind to recombinant GST-PIK3IP162-168 protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot.This antibody failed to react with E.coli lysates and glutathione S transferase （GST）.At the same time,endogenous PIK3IP1 was not detected using 5C6 antibody.Immunofluorescecence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner.CONCLUSION：Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated,which could be utilized as a useful reagent for the analysis of biochemical,structural,and functional properties of PIK3IP1.