| 标记蛋白HPT的表达、纯化及免疫活性鉴定 |
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| 引用本文： | 高彬,张海燕,范海.标记蛋白HPT的表达、纯化及免疫活性鉴定[J].植物研究,2011,31(1):56-60. |
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| 作者姓名： | 高彬 张海燕 范海 |
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| 作者单位： | 1.山东师范大学逆境植物重点实验室，济南 250014；;2.中国科学院植物研究所，北京 100093 |
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| 基金项目： | 国家“863”计划(2007AA091701) |
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| 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。
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| 关 键 词： | 潮霉素磷酸转移酶 原核表达 重组蛋白 多克隆抗体 |
Expression,Purification and Immunoreactivity of Hygromycin-B-Phosphotransferase |
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| GAO Bin, ZHANG Hai-Yan FAN Hai.Expression,Purification and Immunoreactivity of Hygromycin-B-Phosphotransferase[J].Bulletin of Botanical Research,2011,31(1):56-60. |
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| Authors: | GAO Bin ZHANG Hai-Yan FAN Hai |
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| Institution: | GAO Bin1,2 ZHANG Hai-Yan2 FAN Hai1(1.Key Laboratory of Plant Stress,Shangdong Normal University,Jinan 250014,2.Institute of Botany,the Chinese Academy of Sciences,Beijing 100093) |
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| 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. |
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| Keywords: | hygromycin-B-phosphotransferas prokaryotic expression recombinant protein polyclonal antibodies |
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