【摘要】 目的构建胰岛素样生长因子2mRNA结合蛋白3(IGF2BP3)的原核表达载体,在大肠杆菌中表达并纯化IGF2BP3-His融合蛋白,制备和鉴定小鼠抗人IGF2BP3多克隆抗体。方法用PCR方法扩增IGF2BP3基因,构建到pET-28a原核表达载体中,转化大肠杆菌BL21(DE3),诱导IGF2BP3蛋白的表达。所获得的蛋白经镍柱亲和层析纯化,并用透析方法脱去尿素使蛋白复性,获得IGF2BP3原核表达蛋白。将制备的原核表达蛋白免疫BALB/c小鼠,制备多克隆抗体,再用ELISA、Western blot法、免疫组织化学法对抗体的反应性及特异性进行鉴定。结果成功克隆出IGF2BP3基因,经测序与GenBank公布的序列一致;PCR酶切鉴定证实成功构建了pET-28a-IGF2BP3原核表达载体;质粒在BL21(DE3)中成功表达出相对分子质量(Mr)为70000左右的融合蛋白,纯化后经SDS-PAGE分析纯度在90%以上。ELISA确定抗体效价在1∶50000以上,且Western blot法和免疫组织化学技术均证实多克隆抗体能特异性识别目的蛋白。结论成功制备了特异性的小鼠抗人IGF2BP3的多克隆抗体。更多还原

【Abstract】 Objective To construct the prokaryotic expression vector of insulin-like growth factor 2 mRNA-binding protein3(IGF2BP3),express and purify IGF2BP3 protein in E.coli,and prepare the polyclonal antibody against IGF2BP3.Methods The ful open reading frame(ORF) of human IGF2BP3 was amplified by PCR,subcloned into p ET-28 a vector,and transformed into E.coli BL21(DE3),in which expression of the His-tagged IGF2BP3 protein was induced by IPTG.This protein was subsequently purified by Ni-NTA purification system,refolded by removal of urea from the solution.BALB / c mice were immunized with the purified IGF2BP3 protein to produce polyclonal antibody against IGF2BP3.The resulting anti-sera were further characterized by ELISA,Western blotting and immunohistochemistry.Results IGF2BP3 gene we amplified was consistent with the sequence reported by Gen Bank.Prokaryotic expression vector p ET-28a-IGF2BP3 was constructed.His-tagged IGF2BP3 protein was successfully expressed in BL21(DE3) with relative molecular mass(Mr) about 70 000 after IPTG induction.After purified by Ni-NTA resin,the purity of the protein reached above 90%.After immunization,the titer of the IGF2BP3 mouse anti-serum was over 1 ∶ 50 000 as determined by ELISA.Further,Western blotting and immunohistochemistry showed that the IGF2BP3 antibody could specifically recognize the target protein.Conclusion The polyclonal antibody specifically recognizing IGF2BP3 has been successfully generated.更多还原