论文部分内容阅读
为研究CTCF的功能,进一步探索肿瘤的发病机制,我们成功制备了人转录因子CTCF N端融合蛋白多克隆抗体,并初步用于肿瘤细胞CTCF表达状态的分析。采用已构建的表达质粒PGEX-4T-2-CTCF-N,经IPTG诱导表达GST-CTCF N融合蛋白,亲和层析纯化GST-CTCF N融合蛋白,免疫新西兰大白兔制备多克隆抗体。并通过Western blot,对肿瘤细胞和组织CTCF蛋白表达状态进行初步分析。本实验成功在大肠杆菌BL21中诱导表达了GST-CTCF N融合蛋白,经亲和层析纯化获得了纯化的融合蛋白。免疫新西兰大白兔获得了较高生物活性和特异性的多克隆抗体,Western blot证实此多克隆抗体能够特异性识别HepG2、HeLa、MCF-7癌细胞及组织的内源性CTCF蛋白,为今后从蛋白质水平研究CTCF基因表达调控研究打下了基础。
In order to study the function of CTCF and to further explore the pathogenesis of tumor, we successfully prepared the polyclonal antibody of CTCF N-terminal fusion protein, which was initially used in the analysis of CTCF expression in tumor cells. The constructed expression plasmid PGEX-4T-2-CTCF-N was used to express GST-CTCF N fusion protein by IPTG. The GST-CTCF N fusion protein was purified by affinity chromatography and immunized New Zealand white rabbits to prepare polyclonal antibodies. The expression of CTCF protein in tumor cells and tissues was analyzed by Western blot. In this experiment, we successfully induced GST-CTCF N fusion protein in E.coli BL21 and purified the fusion protein by affinity chromatography. Immunization of New Zealand white rabbits to obtain a higher biological activity and specificity of the polyclonal antibody, Western blot confirmed that this polyclonal antibody can specifically recognize HepG2, HeLa, MCF-7 cancer cells and tissue endogenous CTCF protein for the future from Protein level research CTCF gene expression regulation lay the foundation.