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

作　　者：张婵 张瑞凯 刘雁霞 樊振川 ZHANG Chan;ZHANG Ruikai;LIU Yanxia;FAN Zhenchuan(State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science&Technology,Tianjin 300457,China;International Scientific and Technological Institute of Health Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学食品营养与安全国家重点实验室,天津300457 [2]天津科技大学大健康生物技术国家国际科技合作基地,天津300457

出　　处：《食品与生物技术学报》2024年第9期65-71,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(32070698)。

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∶102400,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。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∶102400,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.

关 键 词：莱茵衣藻 多克隆抗体 ARL3 纤毛 

分 类 号：Q78[生物学—分子生物学]