PHF10磷酸化多克隆抗体的制备与其在胃癌细胞PHF10磷酸化水平检测中的应用

Preparation of phosphorylational polyclonal antibody to PHFIO and its application in detecting phosphorylation status in gastric cancer cells

目的 制备PHD指蛋白 10（PHF10）磷酸化多克隆抗体并用于PHF10蛋白的研究,探讨其在胃癌发生发展中的作用与机制.方法 结构预测后应用固相法合成磷酸化多肽.应用磷酸化多肽免疫家兔得到多克隆抗体,并利用Dotblot方法进行此抗体特异性效价的鉴定,结果显示PHF10最低阳性检测浓度为7.8μg/L.利用PHF10磷酸化多抗对8株胃癌细胞和胃黏膜永生化细胞PHF10蛋白磷酸化水平进行检测,胃癌细胞株PH F10磷酸化水平明显高于胃黏膜永生化细胞GES-1.结果 制备出的PHF10蛋白磷酸化多克隆抗体效价较高,可用于磷酸化PHF10蛋白的鉴定.结论 此多克隆抗体可用于PHF10蛋白的研究.

Objective To prepare the phosphorylational polyclonal antibody to PHD finger protein 10 （PHF10） and apply it to the study on PHF10 in the tumorigenesis and development of primary gastric cancer.Methods Phosphorylated polypeptide of PHF10 was synthesized by GL Biochem Ltd （ Shanghai）.Phosphorylational polyclonal antibody was obtained by immunizing rabbits with the polypeptide.The quality of the antibody was identified by Dot blotting.The minimal detectable concentration of the antibody to PHF10 was 7.8 μg/L.Results The phosphorylational polyclonal antibody to PHF10 was obtained and proved to be effective.By using the phosphorylational polyclonal antibody to PHF10,the phosphorylation status of PHF10 was detected in gastric cancer cells and immortalized gastric epithelial cells,GES-1.The results showed phosphorylation status in PHF10 in gastric cancer cells was higher than that in immortalized gastric epithelial cells,GES-1.Conclusion The phosphorylational polyclonal antibody could be applied to the research on phosphorylation status in PHF10 and phosphorylated PHF10 was expressed higher in gastric cancer cells than in GES-1 but varied in intensity.