着丝粒相关蛋白E的抗体制备

Preparation and detection of centromere-associated protein E antibody

目的制备着丝粒相关蛋白E（CENP-E）的兔源特异性多克隆抗体。方法应用分子克隆技术构建原核细胞表达质粒pHis-CENPEC410，并将其转化至表达菌株BL-21（DE3）感受态细胞中；然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达His-CENPEC410融合蛋白，再用Ni-NTA镍离子金属螯合树脂柱亲和层析法进行纯化；最后将纯化后的蛋白作为抗原免疫新西兰大白兔，制备特异性CENP-E多克隆抗体，分别用免疫印迹法和免疫共沉淀法对免疫抗体血清进行检测，用免疫荧光染色法检测纯化的抗体。结果制备的抗体血清可较好地应用于免疫印迹和免疫共沉淀实验，纯化的抗体可用于免疫荧光染色。

Objective To prepare centromere-associated protein E（CENP-E）polyclonal antibody with specificity by using New Zealand white rabbits. Methods Prokaryotic expression plasmid of pHis-CENPEC410 was constructed by molecular cloning technique and then transformed into competent cells of E.coli BL-21 （DE3）. His- CENPEC410 fusion protein was induced by isopropyl β-D-thiogalactoside （IPTG） and purified by affinity chromatog- raphy using Ni-NTA beads. The purified protein was used as antigen to immune New Zealand white rabbits to produce specific polyclonal antibody of CENP-E. The antibodies serum was detected by immunoblotting and co-immunopre- cipitation, and the purified antibodies were detected by immunofluorescene staining. Results The results of immunoblotting and co-immunoprecipitation demonstrated that the antibody serum was effective and the purified antibody could be applied to immunofluorescene test. Conclusions CENP-E polyclonal antibody with high speci- ficity and sensitivity was obtained, which lay the foundation for the follow-up study of CENP-E.