P^(53)融合蛋白的原核表达及其单克隆抗体的制备

STUDY ON APOPTOSIS OF HUMAN HEPATOMA CELLS (BEL 7402) INDUCED BY NORCANTHARIDIN

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。

Objective\ We tried to probe the antitumor mechanisms of new antitumor drug norcantharidin(NCTD) on human hepatoma (BEL 7402) cells. Methods\ By morphological observation and DNA agarose gel electrophoresis, we examined the characteristics of NCTD on human hepatoma cells, then determined the expression of oncoprotein Bcl 2 by immunocytochemical and Western blotting analysis in this process. Results\ Treated with 10mg/L NCTD for 24h, some human hepatoma cells condensed with membrane budding, the nuclear chromatin compaction and membrane enclosed apoptotic bodies formation, and fragments that were multiples of units comprising about 200 base pairs were detected by agarose gel electrophoresis, a characteristic ladder being evident when ethidium bromide stained gels were viewed in ultraviolet light. The expression of oncoprotein Bcl 2 was proved decreasing in human hepatoma cells at this time. Conclusion\ The new antitumor drug NCTD can induce apoptosis in human hepatoma cells, and the declining expression of oncoprotein Bcl 2 might involve in this process.