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P53融合蛋白的原核表达及其单克隆抗体的制备 |
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刘彩云 寿成超 孙素莲 张蕾 曾荔
【摘要】 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。
【关键词】 P53融合蛋白 单克隆抗体 免疫组织化学
EXPRESSION OF P53 FUSION PROTEIN IN PROKARYOTE AND
PREPARATION OF MONOCLONAL ANTIBODY TO P53
Liu CaiyunΔ, Shou Chengchao, Sun Sulian, Zhang Lei, Zeng Li*
(The School of Oncology, Beijing Medical University, Beijing Institute for Cancer Research, Beijing;*Institute of Urology, Beijing Medical University,Beijing)
【Abstract】 Objective The aim in this paper was to prepare anti-P53 monoclonal antibody, which can specifically bind to human P53, and made it widely useful in P53 detection by immunohistochemistry(IHC). Methods The P53 DNA fragment encoding N-terminal 180 amino acid was obtained by PCR and was cloned into PG EX-2T plasmid expressing glutathione S-transferase(GST); The P53-GST fusion proteins expressed by JM109 was used for immunizing BALB/c mice. Results We have raised one hybridoma strain secreting McAb to human P53 (named M126); the IHC analysis of 52 praffin-embedded sections from human breast cancer with M126 and PAB 1801 has shown that the positive immunoreactions were shown in 25 cases (48.1%) and 22 cases (42.3%) respectively; the staining of M126 was stronger and preferable to import P53 McAb PAB 1801. Conclusion M126 can be used instead of PAB 1801 for studying immunohistochemical analysis on P53 protein.
【Key words】 P53 fusion protein; Monoclonal antibody; Immunohistochemistry
野生型P53基因是一种抑癌基团，编码53kD的核蛋白，在正常细胞中参予细胞周期的负调节而控制细胞的增殖［1[1] [2] 下一页
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