抗小鼠干扰素调节因子7多克隆抗体的制备及鉴定

Preparation and identification of polyclonal antibodies against mouse interferon regulatory factor 7

目的获得小鼠干扰素调节因子7(IRF7)抗原,制备抗小鼠IRF7多克隆抗体并鉴定。方法构建原核表达质粒pET‐28a(+)‐IRF7,在大肠杆菌中表达IRF7,用Ni柱纯化获得抗原后,分别采用MnJ胶体锰佐剂和弗氏佐剂制备免疫原,3次免疫后心脏取血分离血清,采用间接ELISA法测定血清效价、纯化试剂盒纯化抗体、Western印迹和免疫沉淀对抗体生物学活性进行鉴定。结果在大肠杆菌中成功表达IRF7并获得纯蛋白。两种佐剂制备的多抗血清,效价均可达到810×10^(3)倍。血清纯化后获得抗IRF7抗体,经Western印迹鉴定,两个多克隆抗体均能够特异识别真核表达的IRF7蛋白,并在免疫沉淀外源表达IRF7时,其效果明显优于商业化抗体。结论成功制备了抗IRF7多克隆抗体,为后续研究IRF7功能及作用机制奠定了基础。

Objective To obtain the protein of recombinant interferon regulatory factor 7(IRF7)of mice,and prepare and identify anti‐IRF7 polyclonal antibodies.Methods The pET‐28a(+)‐IRF7 plasmid was constructed,IRF7 was expressed in E.coli and purified with Ni sepharose.Rabbits were immunized by the purified protein with MnJ and Freund′s adjuvant respectively.The antisera were collected and the titers of anti‐IRF7 antibodies were determined by indirect ELISA.The antibodies were purified by Melon?Gel Monoclonal IgG Purification Kit.The biological activities of the antibodies were assessed by Western blotting and immunoprecipitation.Results The IRF7 protein was purified by Ni sepharose.The titers of polyclonal antibodies prepared by the two adjuvants were 1∶810×10^(3).The polyclonal antibodies were obtained by the antibody purification kit.The eukaryotically expressed IRF7 could be specifically identified by the two polyclonal antibodies,and these antibodies could be used in immunoprecipitation assay.The two polyclonal antibodies were significantly better at precipitation effect than commercial ones.Conclusion The polyclonal antibodies against IRF7 are successfully prepared,which can contribute to studies on the functions of IRF7 and related mechanisms.