原癌基因Zbtb7A的POZ区在大肠杆菌中的表达及其多抗制备

Expression of the POZ domain of human Zbtb7A and preparation of its polyclonal antibodies

目的:原癌基因Zbtb7A在肿瘤的发生发展过程中起"分子开关"的作用。本研究制备Zbtb7A蛋白POZ功能区重组蛋白及相应的多克隆抗体。方法:根据大肠杆菌的密码子偏好性,优化其编码序列,用2步法PCR合成目的片断,并将其构建入原核表达载体pET-26b(+)中。重组表达质粒转入大肠杆菌BL21(DE3),用0.5 mmolIPTG诱导重组蛋白表达。所获重组表达蛋白经15%SDS-PAGE分离,切胶回收目的蛋白,研磨后免疫家兔制备抗血清。收集的抗血清经饱和硫酸铵沉淀和G蛋白柱联合纯化,用Western blot证实对纯化的抗血清进行鉴定。结果:获得了密码子优化后的Zbtb7APOZ区的编码序列,并成功地获得了其重组蛋白,制备了高纯度的抗POZ区多克隆抗体。结论:所获得的重组POZ区蛋白及其多克隆抗体可应用于对Zbtb7A的进一步研究。

Objective： Proto-oncogene Zbtb7A has been characterized as a molecular switch in the process of cancer initiation and development. Our goal is to obtain the POZ domain of the Zbtb7A protein and prepare its polyclonal antibodies. Methods： We optimized the coding sequence of the POZ domain according to the codon bias of E. coli and synthesized the sequence with two-step PCR, which was then introduced into the pET-26b（ ＋ ） vector to express the protein. The recombinant protein was analyzed by 15% SDS-PAGE and the corresponding band was cut out from gel. The minced gel slice that contained the POZ domain protein was injected to immunize rabbits. The collected rabbit antiserum was purified using the saturated ammonium sulfate method in combination with protein G antibody purification, and the purified polyclonal antibodies was evaluated by Western blot. Results： The optimized sequence of the POZ domain was correctly obtained and successfully constructed into the pET-26b（ ＋ ） vector. After induction, an expected protein band about 14 KD was detected on 15% SDS-PAGE, and highly purified polyclonal antibodies were obtained, which were specifically bound to human Zbtb7A,Conclusion： The obtained recombinant protein of the POZ domain and its polyclonal antibodies can be used for further studies of Zbtb7A.