---

作 者 ：高彬;张海燕;范海*

期 刊 ：植物研究 2011年 31卷 1期 页码：56-60

关键词：潮霉素磷酸转移酶；原核表达；重组蛋白；多克隆抗体；

Keywords:hygromycin-B-phosphotransferas, prokaryotic expression, recombinant protein, polyclonal antibodies,

摘 要 ：构建含有HPT的原核表达质粒，经诱导表达纯化后获得HPT抗体。从含有hpt的质粒pCambia1301上扩增目的基因，经SalⅠ和NotⅠ双酶切后与原核表达载体pET-28b（+）进行粘性末端连接，成功构建了pET-28b-hpt质粒，并转化到宿主细菌大肠杆菌Rosset(DE3)中进行蛋白表达。在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下,pET-28b-hpt原核表达载体在E.coli Rosset菌株中以包涵体的形式高效表达了HPT蛋白，并以纯化的目的蛋白为抗原免疫兔制备了多克隆抗体。利用本方法可以获得特异性强、灵敏度高的多克隆抗体。

Abstract:The prokaryotic expression plasmid of hygromycin-B-phosphotransferase(HPT) was constructed. The hpt gene was cloned by PCR. It was digested by SalⅠ/NotⅠand subcloned into expression vector pET-28b(+). pET-28b-hpt was transferred into E. coli Rosset; fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The most products existed in an inclusion body form. The HPT protein purified by Ni²⁺-NTA column was used to immunize New Zealand rabbits. The HPT polyclonal antibodies reveal high sensitivity and specificity.

---