PHF10融合蛋白表达、抗体制备及其在胃癌中的表达的检测

Preparation of PHF10 antibody and analysis of PHF10 expression gastric cancer tissues

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。

AIM： To prepare PHF10 antibody and check the expression of PHF10 protein in the tissues of gastric cancer and adjacent tissue.METHODS： His-tagged PHF10 was expressed in E.coli BL21.Rabbit PHF10 polyclonal antiserum was generated by injecting the purified recombinant His-tagged PHF10 inclusion body as the antigen,and further separated by affinity purification.To confirm the specificity of the PHF10 antibody,transiently expressed Flag-PHF10 fusion protein was analyzed by immunoblotting with anti-flag monoclonal antibody control.The produced antibody Was used to check the expression of PHF10 protein in gastric cancer and adjacent tissues by Western blot.RESULTS： Antibodies specifically binding to PHF10 could be obtained by immunization,and expression of PHF10 was significantly higher in gastric cancerous tissues comparing with adjacent normal tissues and GES-1 shows more PHF10 expression than gastric cancer cell lines with the generated antibody.CONCLUSION： The specific anti-PHF10 antibody is obtained and it could be used to detect the expression of PHF10 protein in gastric cancer cell lines and tissues,in which PHF10 is unregulated in gastric cancer.