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目的为研制出能特异与Ｐ５３结合的单克隆抗体，使对Ｐ５３的检测得到更为广泛的应用。方法用ＰＣＲ技术扩增编码人Ｐ５３Ｎ－端１８０个氨基酸的ＤＮＡ片段并将其克隆在谷胱苷肽转移酶（ＧＳＴ）表达质粒ＰＧＥＸ－２Ｔ中。用该重组质粒转化大肠杆菌ＪＭ１０９，并经异丙基β－Ｄ硫代半乳糖苷（ＩＰＴＧ）诱导产生Ｐ５３－ＧＳＴ融合蛋白。用Ｐ５３－ＧＳＴ免疫ＢＡＬＢ／ｃ小鼠。常规细胞融合，间接酶联免疫吸附试验（ＥＬＩＳＡ）双筛和免疫组织化学（ＩＨＣ）筛选。结果获得一株能稳定分泌抗Ｐ５３单抗的杂交瘤细胞株，其分泌的单抗（命名为Ｍ１２６），亚类为ＩｇＧ２ｂ。用单抗Ｍ１２６与常用Ｐ５３进口单抗ＰＡＢ１８０１（ＺＹＭＥＤ公司）同时对５２例乳腺癌的石蜡切片标本进行ＩＨＣ分析，阳性检测率分别为４８．１％（２５／５２）和４２．３（２２／５２），与ＰＡＢ１８０１相比Ｍ１２６表现出更强的反应特异性，在一定程度上优于ＰＡＢ１８０１。结论Ｍ１２６可替代进口单抗ＰＡＢ１８０１用于Ｐ５３免疫组织化学研究。
The purpose is to develop a monoclonal antibody that binds specifically to P53, making the detection of P53 more widely available. Methods A 180 amino acid DNA fragment coding for the human P53 N-terminal gene was amplified by PCR and cloned into the PGST-expressing plasmid pGEX-2T. Escherichia coli JM109 was transformed with this recombinant plasmid and P53-GST fusion protein was induced by isopropyl β-D thiogalactoside (IPTG). BALB / c mice were immunized with P53-GST. Conventional cell fusion, indirect enzyme-linked immunosorbent assay (ELISA) dual screening and immunohistochemistry (IHC) screening. Results A hybridoma cell line stably secreting anti-P53 mAb was obtained, which secreted monoclonal antibody (named as M126) with subclass IgG2b. IHC analysis of paraffin-embedded specimens from 52 breast cancer patients with monoclonal antibody M126 and the commonly used P53 monoclonal antibody PAB1801 (ZYMED) showed positive rates of 48.1% (25/52) and 42.3 (22 / 52), M126 showed stronger reaction specificity than PAB1801, to a certain extent superior to PAB1801. Conclusion M126 can replace imported monoclonal antibody PAB1801 for P53 immunohistochemistry.