Nono蛋白的原核表达、纯化和多克隆抗体的制备

Prokaryotic Expression,Purification of Nono and Preparation of Its Polyclonal Antibody

目的表达和纯化带多聚组氨酸(6×His)标签的Nono(non-POU-domain-containing,octamer-bindingprotein)融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a(+)-Nono重组表达质粒,转入Rosetta(DE3)大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB/C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a(+)-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。

Objective To express and purify the fusion protein of His-tagged non-POU-domaincontaining, octamer-binding protein （Nono） in prokaryocytes and prepare mouse anti-Nono polyclonal antibody. Methods The expression vector pET-28a（ ＋ ）-Nono was reconstructed and transformed into Rosetta （DE3）. 6 x His-tagged Nono fusion protein was induced by IPTG. The His-Nono fusion protein purified by Ni-NTA His Bind resin was used to immunize BALB/C mouse. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blotting. Results The His-Nono fusion protein was highly expressed in E. coli and specific poly- clonal antibody was obtained after the immunization. Conclusion The recombinant prokaryotic expression vector pET-28a（ ＋ ）-Nono was successfully constructed and high expression of Nono was induced in E. coll.