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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 against spindlin 1 and lay the foundation for functional studies and tumor screening of spindlin 1. Methods spindlin 1-GST was expressed in E. coli. After GST was excised, the purified spindlin 1 protein was immunized in rabbits to prepare polyclonal antibody. Hitrap Protein A column was used to purify spindlin 1 antiserum and ELISA to determine antibody titer. The specificity of the antibody was detected by Western blotting and immunohistochemistry. Results The results of ELISA showed that the titer of purified spindlin 1 polyclonal antibody was 1: 2000. The results of Western blotting showed that the purified spindlin 1 polyclonal antibody could bind to spindlin 1 in HeLa cell extract transfected with pAdeasy-myc-spindlin 1, which was consistent with the detection of Myc. The results of immunohistochemistry showed that HeLa cells transfected with enhanced green fluorescent protein (EGFP) and spindlin 1 (green fluorescent protein (EGFP) and spindlin 1) expressed green fluorescence with spindlin 1 protein in the nucleus. And spindlin 1 polyclonal antibody can specifically recognize ovarian cancer tissue spindlin 1 protein. Conclusion The polyclonal antibody of tumor-associated protein spindlin 1 was successfully prepared and could detect the expression of spindlin 1 in cells and tissues.