人抗P53蛋白单克隆抗体的制备及评价

Preparation and application of a monoclonal antibody against P53 protein of human

目的制备获得人P53蛋白单克隆抗体,并应用于免疫组化检测。方法利用基因重组方法,将p53基因构建到原核质粒载体上并诱导表达,将收集获得的P53蛋白免疫小鼠,经细胞融合及亚克隆手段获得鼠源单克隆抗体,与商品化的抗体DO-1共同染色蜡块包埋的病理组织样本,比较二者的染色结果。结果在筛选获得的与DO-1检测结果相近的5株抗体内,取检测结果最接近的1株抗体5E10,经Protein A/G株纯化抗体,与收集获得的数例癌症组织蜡块进行免疫组化验证,发现结果均与DO-1的结果无明显差异。结论成功制备并筛选出1株人抗P53单克隆抗体,可用于免疫组化染色。

The study performed to prepare a monoclonal antibody against human P53 protein, and evaluate its application in immunohistochemistry. The p53 gene was constructed into prokaryotic expression plasmid by gene recombination methods. P53 protein was expressed in E.coil, and then used to immunize Balb/c mice. P53 monoclonal antibody was prepared by using cell fusion and subcloning methods. Using the mAb and DO-l, IHC and specificity experiments were carry out on wax block organizations collected from hospital. Five antibodies against P53 protein were obtained, and one of them demonstrated most close quality to DO-l, named as 5El0. After purification by protein A/G. the McAbs （5El0） was used for immunohistochemistry and specificity experiments on primary colon carcinoma slice, which demonstrated no significant different as compared with the detection using DO-1. In conclusion, one monoclonal antibody （5E10） against P53 protein of human has prepared, which could be used in the IHC detection for human P53 protein.