莱茵衣藻小G蛋白ARL1的原核表达、纯化及多克隆抗体制备  

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Small G Protein ARL1 of Chlamydomonas reinhardtii

作　　者：陶冶 鲍冬雪[1,2] 刘雁霞 樊振川 TAO Ye;BAO Dongxue;LIU Yanxia;FAN Zhenchuan(State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science&Technology,Tianjin 300457,China;National International Science and Technology Cooperation Base for Big 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：《天津科技大学学报》2024年第6期14-20,共7页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金面上项目(32070698);国家自然科学基金青年科学基金项目(32200558)。

摘　　要：为研究小G蛋白ARL1在纤毛生成和纤毛信号转导过程中的作用,特制得一种特异性较强的莱茵衣藻(Chlamydomonas reinhardtii)ARL1多克隆抗体。构建6×His标签的原核表达载体pET-28a(+)-arl1,并在大肠杆菌(E.coli)DH5α/C+感受态中诱导表达融合蛋白6×His-ARL1,经纯化后免疫新西兰雄性大白兔。3次免疫后取耳动脉血清,以间接ELISA法测试效价后取血。通过Protein A和抗原抗体亲和纯化后获得了灵敏度高、特异性强的ARL1抗体。通过免疫荧光和免疫印迹(Western blot)检测,发现大量ARL1定位于细胞质、细胞基体和纤毛,为后续研究ARL1在莱茵衣藻纤毛生成和信号转导中的功能奠定了基础。In order to study the role of small G protein ARL1 in ciliary formation and ciliary signal transduction,a highly specific polyclonal antibody against ARL1 of Chlamydomonas reinhardtii was prepared.First,6×His labeled prokaryotic expression vector pET-28a(+)-arl1 was constructed,and the fusion protein 6×His-ARL1 was induced in E.coli DH5α/C+,and then purified and immunized New Zealand male white rabbits.After three times of immunization,the serum of auricular artery was taken,and the titer was tested by indirect ELISA.ARL1 antibody with high sensitivity and specificity was obtained by affinity purification of Protein A and antigenic antibody.By immunofluorescence and Western blot detection,ARL1 was found to be localized in cytoplasm,basal body and cilia,which has laid a foundation for further study on the function of ARL1 in cilia generation and signal transduction of C.reinhardtii.

关 键 词：ARL1 原核表达 多克隆抗体 莱茵衣藻 

分 类 号：Q933[生物学—微生物学]