针对人Tudor-SN蛋白T103位点的应激磷酸化抗体制备及分析

目的：针对人Tudor-SN蛋白T103位点（103位苏氨酸,Thr103）制备兔源多克隆磷酸化抗体,并进行应激磷酸化的时相分析。方法：首先人工合成含磷酸化T103（pT103）位点的多肽,4次免疫新西兰大白兔后获取抗血清;然后以AKTA蛋白纯化系统进行纯化,并利用Western blotting和细胞免疫荧光实验对纯化后的抗pT103抗体进行鉴定;最后以In-cell Western法进行Tudor-SN蛋白的应激磷酸化/去磷酸化时相性分析。结果：（1）确定并合成磷酸化多肽“TIENKpTPQGRC”,收集约75 ml兔源抗血清,纯化后获取2.08 mg/ml抗pT103抗体;（2）当HeLa细胞受到氧化应激时,以pT103抗体检测的磷酸化信号增强,可在胞浆中检测到颗粒状信号,与内源性Tudor-SN应激颗粒存在共定位关系;（3）在氧化应激及应激去除后恢复过程中,T103位点的磷酸化水平呈现一定的波动性时相。结论：成功制备针对Tudor-SN蛋白T103位点的兔源多克隆抗pT103抗体,有助于从磷酸化修饰角度进行Tudor-SN在细胞应激方面的机制探讨。

The Analysis and Preparation of the Stress Associated Phosphorylation Antibodies Specific for the T103 Site of Tudor-SN Protein

Objective: To prepare and analyze the rabbit polyclonal stress associated phosphorylation antibodies specific for the T103 (Thr103) site of Tudor-SN protein. Methods: The phosphorylated T103 (pT103) site-containing polypeptide was synthesized; Antiserums were collected from the New Zealand white rabbits immunized with the polypeptide four times and purified through AKTA protein purification system. The Western blotting analysis,immunofluorescence experiment and In-cell Western assay were performed using the purified anti-pT103 antibodies. Results: (1) The phosphorylated peptide "TIENKpTPQGRC" was synthesized and about 75 ml rabbit antiserums were collected (2.08 mg/ml); (2) The phosphorylation signal was detected and increased under oxidative stress conditions in Western blotting analysis; the granule signal was also observed in the cytoplasm of HeLa cells and co-localized with the endogenous Tudor-SN-labeled stress granule during stress. (3) The phosphorylation level of Tudor-SN at T103 site fluctuates during the oxidative stress and after the removal of stress. Conclusion: The rabbit polyclonal anti-pT103 antibodies specific for the T103 site of Tudor-SN were prepared successfully. It will help to study the role of Tudor-SN phosphorylation modification in cellular stress responses.