肿瘤相关蛋白spindlin 1的表达、纯化及多克隆抗体的制备(英文)

Expression and purification of spindlin 1,a novel cancer related protein,and preparation of its polyclonal antibody

目的制备spindlin 1蛋白的多克隆抗体,为spindlin 1的功能研究和肿瘤筛查奠定基础。方法 spindlin 1-谷胱甘肽S转移酶融合蛋白(spindlin 1-GST)在大肠杆菌中表达,切除GST后,将纯化的spindlin 1蛋白免疫家兔,制备多克隆抗体。Hitrap Protein A柱纯化spindlin 1抗血清,ELISA测定抗体效价。用Western蛋白质印迹法和免疫组化方法检测抗体的特异性。结果 ELISA检测结果表明,纯化的spindlin 1多克隆抗体效价为1∶2000。Western蛋白质印迹法实验结果表明,纯化的spindlin 1多克隆抗体能够与转染pAdeasy-myc-spindlin 1载体的HeLa细胞抽提物中spindlin 1蛋白结合,与Myc抗体检测位置一致。免疫组化结果表明,转染增强型绿色荧蛋白(EGFP)和spindlin 1(pEGFP-C3-spindlin 1)的HeLa细胞发绿色荧光,其细胞核中有spindlin 1蛋白表达。且spindlin 1多克隆抗体能够特异性识别卵巢癌组织中的spindlin 1蛋白。结论成功制备肿瘤相关蛋白spindlin 1的多克隆抗体,能够特异性检测细胞和组织中spindlin 1蛋白表达。

OBJECTIVE To prepare a polyclonal antibody for spindlin 1 protein,a novel cancer related protein,and to provide the data for a better understanding of its functions and screening tumor. METHODS Purified spindlin 1 protein was injected into rabbits to produce the polyclonal antiserum after removing glutathione S-transferase（ GST） from the fusion protein spindlin 1-GST that was expressed in Escherichia coli.. The antiserum was purified through the Hitrap Protein A system,and the titer of spindlin 1 polyclonal antibody was detected by ELISA. The specificity of the polyclonal antibody was determined by Western blotting and immunohistochemistry. RESULTS The titer of spindlin 1 polyclonal antibody was 1∶ 2000. Western blotting detection demonstrated that the spindlin 1 polyclonal antibody recognized myc-spindlin 1 recombinant fusion protein in HeLa cells transfected with pAdeasy-myc-spindlin 1, which also corresponded with Myc. antibody. The HeLa cells were transfected with enhanced green fluorescence protein（ EGFP） and spindlin 1 vector（ pEGFP-C3-spindlin 1）,which was confirmed by the independent GFP fluorescence assay. The results of immunohistochemistry detection with the spindlin 1 polyclonal antibody suggested that spindlin 1 was mainly expressed in the nuclei of HeLa cells. More importantly,in immunohistochemical assays,the spindlin 1 antibody recognized nuclear spindlin 1 expression in clinical ovarian cancer tissues. CONCLUSION The specific spindlin 1 polyclonal antibody is prepared,which may be used to detect cancer-related protein spindlin 1 in HeLa cells and ovarian cancer tissues.