果蝇Domeless蛋白的原核表达及多克隆抗体的制备研究  

Prokaryotic Expression of the Drosophila Domeless Fusion Protein and Preparation of Its Polyclonal Antibody

作　　者：唐超[1] 任恋[1] 刘宪楚[1] 李容[1] 江志钢[1] 赵阳[1] 廖四芳[1] 袁佳佳[1] 莫小阳[1] 吴秀山[1] 

机构地区：[1]湖南师范大学生命科学学院,蛋白质化学及鱼类发育生物学教育部重点实验室,心脏发育研究中心,中国湖南长沙410081

出　　处：《生命科学研究》2013年第2期116-119,124,共5页Life Science Research

基　　金：国家自然科学基金资助项目(30930054)

摘　　要：在果蝇(Drosophila melanogaster)的研究中发现Domeless接受器参与发育期间的JAK/STAT信号调节,在心脏疾病的发生机制中发挥重要作用.为了克隆Domeless,我们利用生物信息学选择果蝇Domeless基因抗原亲水区,将扩增出的PCR片段克隆到原核表达pET-28a载体中,转入E.coli(Escherichia coli)中后通过IPTG(Isopropylβ-D-thiogalactoside)诱导融合蛋白表达,Ni-IDA凝胶柱亲和纯化,纯化后的His-Domeless融合蛋白免疫新西兰大白兔制备多克隆抗体.用Western blot检测抗体的效价和特异性.获得了Domeless原核表达重组融合蛋白以及高效价的、特异性兔抗Domeless多克隆抗体,为后续Domeless功能研究奠定了基础.The Drosophila Domeless receptor is reported to be involved in the JAK/STAT signaling pathway during the development, and have an important role in pathogenic mechanisms of cardiovascular diseases. In order to clone Dorneless, the hydrophilie antigen region of Domeless was identified by bioinformatic method. Domeless fragment was amplified by PCR and purified. The fragment was inserted into the prokaryotic expression vector pET-28a. This new recombined plasmid was transformed into E.coli and expression of the GST fusion protein was induced with IPTG. After purification by Ni-IDA gel column affinity chromatography, His-Dorneless fusion protein was used to immune the New Zealand white rabbits to prepare polyclonal antibody. The antibody titer and specificity was tested by Western blot. GST-tag fusion protein of Domeless and anti-Domeless polyclonal antibody with high sensitivity and specificity were successfully obtained, which can laid foundation for the further studies of Domeless function .

关 键 词：Domeless 果蝇 原核表达 多克隆抗体 

分 类 号：Q71[生物学—分子生物学]