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目的:制备FHL1及其剪接体FHL1B、FHL1C的多克隆抗体,并对其特异性进行鉴定。方法:融合表达GST-FHL1蛋白,常规免疫小鼠,制备多克隆抗体。分别构建带有FLAG标签的FHL1-5、FHL1B、FHL1C以及FHL1N端和C端缺失突变体的真核表达载体。Westernblot检测抗体的特异性。结果:获得了FHL1-5、FHL1B、FHL1C及FHL1(1-169aa)和FHL1(168-280aa)的真核表达载体;得到的多克隆抗体可特异识别FHL1、FHL1B和FHL1C,而不识别FHL2-5;抗体与FHL1N端反应,而不与其C端反应。结论:成功得到可特异识别FHL1、FHL1B和FHL1C的多克隆抗体,为进一步研究FHL1及其剪接突变体的功能奠定了坚实的基础。
Objective: To prepare polyclonal antibodies to FHL1 and its spliceosomes FHL1B and FHL1C and to identify its specificity. Methods: Polyclonal antibody was prepared by immunizing mice with GST-FHL1 fusion protein. The FLAG-tagged FHL1-5, FHL1B, FHL1C and FHL1N-terminal and C-terminal deletion mutants were constructed respectively. Antibodies were detected by Western blot. Results: The eukaryotic expression vectors of FHL1-5, FHL1B, FHL1C, FHL1 (1-169aa) and FHL1 (168-280aa) were obtained. The obtained polyclonal antibodies could specifically recognize FHL1, FHL1B and FHL1C without recognizing FHL2- 5; the antibody reacts with the FHL1 N terminus without reacting with its C terminus. Conclusion: The polyclonal antibodies that can specifically recognize FHL1, FHL1B and FHL1C were successfully obtained, which laid a solid foundation for further study on the function of FHL1 and its splice variants.