小G蛋白ARL3多克隆抗体制备及其突变体鉴定

Preparation of Polyclonal Antibodies Against the Small G Protein ARL3 and Identification of Its Mutants

摘要:

为探究ARL3在纤毛生成和纤毛信号转导中的功能，制备了兔抗莱茵衣藻ARL3多克隆抗体，并鉴定了arl3突变体。通过基因克隆获取arl3目的基因，构建原核表达载体pET-28a（+）-arl3，以大肠杆菌BL21（DE3）为宿主表达融合蛋白6×His-ARL3。将纯化后的目的蛋白质用于免疫兔子并获得抗血清，随后对抗血清中的protein A和抗原抗体进行纯化，并通过蛋白质免疫印迹检测莱茵衣藻ARL3抗原。结果表明，抗血清效价可达1∶102 400，纯化后的ARL3抗体在1∶400的稀释条件下检测出单一目的条带。表明作者成功制备了高特异性、高灵敏度的兔抗莱茵衣藻ARL3蛋白多克隆抗体，且鉴定出一株arl3突变体，为进一步研究莱茵衣藻ARL3蛋白在纤毛内的功能提供了基础。

Abstract:

To investigate the function of ARL3 in ciliogenesis and ciliary signal transduction, the author generate a rabbit polyclonal antibody against Chlamydomonas reinhardtii ARL3 and to identify arl3 mutants. The target gene, arl3, was obtained through gene cloning, and a prokaryotic expression vector, pET-28a(+)-arl3, was constructed for expression in Escherichia coli BL21 (DE3) to produce the fusion protein 6×His-ARL3. The purified target protein was used to immunize rabbits, resulting in the production of antiserum. The antiserum was subsequently purified using protein A and antigen-antibody, and Western blot was employed to detect the presence of the Chlamydomonas reinhardtii ARL3 antigen. Results indicated that the antiserum titer reached 1∶102 400, with the purified ARL3 antibody detecting a single specific band at a dilution of 1∶400. This confirms the successful preparation of a highly specific and sensitive rabbit polyclonal antibody against Chlamydomonas reinhardtii ARL3, as well as the identification of an arl3 mutant. These findings provide a foundational basis for further investigation into the function of ARL3 protein within cilia.