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目的:制备PHF10多克隆抗体,并检测胃癌细胞系和胃黏膜永生化细胞株GES-1中PHF10表达谱。方法:在大肠杆菌BL21中表达GST标签标记的PHF10融合蛋白,以纯化的PHF10重组融合蛋白为抗原制备兔PHF10多抗血清。进一步利用亲和纯化方法制备PHF10多克隆抗体;为确定PHF10抗体的特异性,通过Western blot检测瞬时表达的Flag-PHF10融合蛋白,同时以抗Flag单克隆抗体(mAb)作为对照;利用制备的抗体通过Western blot检测PHF10在GES-1和各种胃癌细胞系中的表达。结果:与PHF10特异性结合的抗体能够通过免疫法获得;利用制备的抗体检测到PHF10在各胃癌细胞系与GES-1之间表达差异有统计学意义。结论:获得了特异性抗PHF10抗体,用该抗体可以检测到胃癌细胞系中PHF10的表达,可能是一个新的肿瘤靶标。
OBJECTIVE: To prepare PHF10 polyclonal antibody and to detect PHF10 expression in gastric cancer cell line and gastric mucosa immortalized cell line GES-1. Methods: GST-tagged PHF10 fusion protein was expressed in E. coli BL21, and purified PHF10 recombinant fusion protein was used as antigen to prepare rabbit PHF10 polyclonal antiserum. In order to determine the specificity of PHF10 antibody, the transient expression of Flag-PHF10 fusion protein was detected by Western blot and the anti-Flag monoclonal antibody (mAb) was used as a control; the prepared antibody The expression of PHF10 in GES-1 and various gastric cancer cell lines was detected by Western blot. RESULTS: Antibodies specifically binding to PHF10 could be obtained by immunoassay. There was a significant difference in the expression of PHF10 between gastric cancer cell lines and GES-1 detected by the prepared antibodies. CONCLUSION: The specific anti-PHF10 antibody was obtained, which can be used to detect the expression of PHF10 in gastric cancer cell lines, which may be a new tumor target.