论文部分内容阅读
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。
Objective To construct a prokaryotic expression vector for insulin-like growth factor 2 (IGF2) 3 binding protein 3 (IGF2BP3) and express and purify IGF2BP3-His fusion protein in E. coli to prepare and identify mouse anti-human IGF2BP3 polyclonal antibody. Methods The IGF2BP3 gene was amplified by PCR and inserted into prokaryotic expression vector pET-28a. The recombinant plasmid was transformed into E. coli BL21 (DE3) to induce IGF2BP3 protein expression. The obtained protein was purified by nickel column affinity chromatography, and urea was dialyzed to renature the protein to obtain the prokaryotic expression protein of IGF2BP3. The prepared prokaryotic expression protein was immunized BALB / c mice to prepare polyclonal antibodies, then by ELISA, Western blot, immunohistochemistry to identify the reactivity and specificity of the antibody. Results The IGF2BP3 gene was successfully cloned and sequenced. The PCR product was verified by restriction endonuclease digestion and DNA sequencing. The prokaryotic expression vector pET-28a-IGF2BP3 was constructed successfully. Plasmid was successfully expressed in BL21 (DE3) About 70,000 fusion protein, purified by SDS-PAGE analysis of more than 90% purity. The antibody titer was above 1: 50000 by ELISA, Western blot and immunohistochemistry confirmed that the polyclonal antibody could specifically recognize the target protein. Conclusion The specific mouse anti-human IGF2BP3 polyclonal antibody was successfully prepared.