[Expression of recombinant human dihydropyrimidine dehydrogenase and its application to the preparation of anti-DPD antibodies for immunochemical detection].
[Expression of recombinant human dihydropyrimidine dehydrogenase and its application to the preparation of anti-DPD antibodies for immunochemical detection].
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重组人二氢嘧啶脱氢酶的表达及其在免疫化学检测抗DPD抗体制备中的应用
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
M. Fukushima
中科院分区：
文献类型：
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作者：
H. Okabe;K. Arakawa;T. Takechi;M. Fukushima
二氢嘧啶脱氢酶（DPD）是5-氟尿嘧啶（5-FU）催化的关键酶，并且其在肿瘤中的表达被认为降低5-FU对肿瘤的功效。为了通过免疫印迹和/或免疫组织化学方法检测DPD分子，我们试图制备针对使用hDPD cDNA在杆状病毒表达系统中表达的重组人DPD（rhDPD）的高度特异性抗体。表达的rhDPD蛋白被发现保留其完整的分子形式，并显示出高的5-FU降解活性相当于人肝DPD。使用这种重组蛋白，单克隆和多克隆抗体ehDPD的产生和它们的特异性，酶活性的关系和肿瘤DPD表达的免疫组织化学测量的可能性进行了研究。结果表明，抗rhDPD单克隆抗体仅识别人DPD，而抗rhDPD多克隆抗体与人和啮齿动物的DPD反应。26个肿瘤细胞的DPD含量与5-FU降解活性密切相关（r = 0.874）。此外，使用我们的抗rhDPD抗体对肿瘤细胞DPD表达的免疫组织化学评价显示，表达高水平DPD的肿瘤细胞显示强阳性染色，但不表达低水平或无DPD的肿瘤细胞显示强阳性染色。这些结果表明，使用我们的抗rhDPD抗体的肿瘤DPD表达的免疫化学检测可能是一种手段，以预测临床反应5-FU为基础的化疗。
Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the catabolism of 5-fluorouracil (5-FU), and its expression in a tumor is thought to reduce the efficacy of 5-FU against the tumor. To detect a DPD molecule by immunoblotting and/or immunohistochemical methods, we attempted to prepare highly specific antibodies against recombinant human DPD (rhDPD) expressed in the baculovirus-expression system using hDPD cDNA. The expressed rhDPD protein was found to retain its entire molecular form and to show a high 5-FU-degrading activity equivalent to that of human liver DPD. Using this recombinant protein, both monoclonal and polyclonal antibodies to ehDPD were generated and their specificities, relationship to enzyme activity and the possibility of immunohistochemical measurement of tumoral DPD expression were investigated. The results revealed that anti-rhDPD monoclonal antibodies recognized only human DPD, while anti-rhDPD polyclonal antibodies reacted with both human and rodent DPD. The DPD content in 26 tumor cells, estimated by immunoblotting, was closely related to the 5-FU-degrading activities in those cells (r = 0.874). Moreover, immunohistochemical evaluation of tumor cellular DPD expression using our anti-rhDPD antibodies revealed that tumor cells expressing high levels of DPD showed strongly positive staining, but not those expressing low level or no DPD. These results suggest that immunochemical detection of tumoral DPD expression using our anti-rhDPD antibodies may be a means to predict the clinical response to 5-FU-based chemotherapy.