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目的:构建人SUMO-2基因的原核表达载体,纯化融合蛋白GST-SUMO2-SUMO2并以其为抗原免疫家兔,制备人SUMO-2多克隆抗体。方法:用PCR的方法得到人SUMO-2基因并克隆至pET41a(+)原核表达载体中,转化大肠杆菌BL21(DE3)plysS诱导融合蛋白GST-SUMO2-SUMO2表达;所获得的可溶性蛋白经亲和层析纯化、SDS-PAGE电泳鉴定后,免疫家兔制备抗血清,分别采用ELISA、Western blot检测抗体效价和特异性。结果:测序证实重组质粒pET41a(+)-SU-MO2-SUMO2构建成功;SDS-PAGE结果证实获得Mr为52000的GST-SUMO2-SUMO2融合蛋白且为可溶性蛋白;经过GST亲和层析有效纯化;以该融合蛋白免疫家兔制备得到的抗血清经Western blot检测证实能与目的蛋白发生特异性结合,ELISA检测为阳性。结论:获得了人SUMO-2蛋白及特异性多克隆抗体,对进一步研究人SUMO-2及SUMO第二类家族的功能提供了有用工具。
OBJECTIVE: To construct the prokaryotic expression vector of human SUMO-2 gene and purify the fusion protein GST-SUMO2-SUMO2 and use it as an antigen to immunize rabbits to prepare human SUMO-2 polyclonal antibody. Methods: Human SUMO-2 gene was cloned by PCR and cloned into pET41a (+) prokaryotic expression vector. The fusion protein GST-SUMO2-SUMO2 was induced by E.coli BL21 (DE3) plysS. After purification by chromatography and identification by SDS-PAGE electrophoresis, rabbits were immunized to prepare antiserum. The antibody titer and specificity were determined by ELISA and Western blot respectively. Results: The recombinant plasmid pET41a (+) - SU-MO2-SUMO2 was successfully constructed and confirmed by SDS-PAGE. GST-SUMO2-SUMO2 fusion protein with Mr 52000 was confirmed as soluble protein and purified by GST affinity chromatography. The antisera prepared from rabbit immunized with the fusion protein was confirmed by Western blot to bind specifically with the target protein, and the ELISA assay was positive. CONCLUSIONS: Human SUMO-2 protein and specific polyclonal antibodies were obtained, which provided a useful tool for further study on the functions of SUMO-2 and SUMO in the second family.