Method for Protein Phosphorylation Analysis by Use of Antibodies Specific to Phosphorylation Motifs

本研究的主要目的是研制用于蛋白质磷酸化分析的“磷酸化基序”特异性单抗。蛋白激酶修饰的磷酸化位点特异性抗体是研究蛋白质磷酸化的重要工具。含有磷酸丝氨酸的磷肽通常被用来作为抗体生产的表位。然而，磷酸丝氨酸在循环过程中容易与磷酸酶结合，从而产生针对非磷酸化肽的特异性抗体。我们试图生产针对肿瘤抑制蛋白P53的Ser6和SE9位点的磷酸肽特异性抗体。当我们用规则的磷酸多肽作为表位免疫兔子时。虽然产生了针对Ser9磷酸化位点的抗体，但没有观察到Ser6磷酸化特异性抗体的产生。为了克服这一问题，合成了稳定的磷酸丝氨酸衍生物L-2-氨基-4，4-二氟丁酸(F2Pab)，并将其掺入多肽中进行免疫。利用含F_2Pab的多肽，我们成功地获得了SER6磷酸化特异性抗体。这一结果表明，用F_2Pab制备磷酸化特异性抗体是有效的。其次，我们建立了磷酸化基序特异性单抗的制备方法。我们选择了由Ser(P)-Gln序列组成的ATM磷酸化基序。抗该基序的单抗只识别含有Ser(P)-Gln序列的磷酸肽，不能与不同磷酸化基序的磷酸肽发生反应。在抗体产生的过程中，我们还发现，短期免疫的淋巴细胞比定期免疫的脾细胞更有效地产生特异性抗体。

The special aim in this study is to develop "phosphorylation motif"-specific monoclonal antibody for analysis of protein phosphorylation. Antibody specific to phosphorylation site modified by protein kinase is an essential tool for the study of protein phosphorylation. Phosphopeptide containing phosphoserine is usually used as epitope for antibody production. However phosphoserine is liable to phosphatase during circulation, resulting in antibody specific to unphosphorylated peptide. We tried to produce phosphopeptide-specific antibodies for the Ser6 and Se9 sites of tumor suppressor protein p53. When we used regular phosphopeptides as epitope to immunize rabbits. Although an antibody against Ser9 phosphorylation site was produced, no production of a Ser6 phosphorylation-specific antibody was observed. To overcome this problem, the stable phosphoserine derivative L-2-amino4-phosphono-4, 4-difluorobutanoic acid (F_2Pab) was synthesized to incorporate into peptides for immunization. Using F_2Pab-containing peptide, we have successfully obtained the Ser6 phosphorylation-specific antibody. This result demonstrates that the method using F_2Pab is effective for phosphorylation-specific antibody production. Second, we have developed the method to produce phosphorylation motif-specific monoclonal antibody. We selected the ATM phosphorylation motif that consists the sequence of Ser(P)-Gln. The monoclonal antibody for the motif only recognized phosphopeptides containing the Ser(P)-Gln sequence and did not react to phosphopeptides with different phosphorylation motifs. During the process of antibody production, we also found that lymphnode cells with short term immunization produced the specific antibody more effectively than spleen cells with regular term immunization.