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作 者:何龙[1] 羊健[2] 张松柏[3] 张恒木[2] 刘勇[1,3] 陈剑平[1,2]
机构地区:[1]湖南农业大学植物保护学院,湖南长沙410128 [2]浙江省农业科学院病毒学与生物技术研究所,浙江杭州310021 [3]湖南省农业科学院植物保护研究所,湖南长沙410125
出 处:《生物技术通讯》2016年第4期525-528,共4页Letters in Biotechnology
摘 要:目的:克隆水稻COP9信号复合物5B亚基(CSN5B)的基因,原核表达CSN5B蛋白并制备多克隆抗体。方法:用RT-PCR技术从日本晴水稻中扩增得到CSN5B蛋白基因Os CSN5B并将其连接至p EASYTM-T5 Zero克隆载体,然后将其亚克隆至原核表达载体p ET-32a+,并导入大肠杆菌BL21plys S宿主菌中诱导表达;重组的CSN5B融合蛋白经Ni-NTA His.Bind Resin纯化后免疫兔子制备多克隆抗体,并经Western印迹分析。结果与结论:获得了CSN5B蛋白的特异性抗体,Western印迹显示CSN5B蛋白在水稻植株中呈高水平表达,为进一步探讨该基因的功能奠定了基础。Objective: To clone a gene encoding 5B subunit of COP9 signal complex(CSN5B) from rice plants,express the CSN5 B protein in prokaryotic system, prepare and apply the antiserum specifically against the CSN5 Bprotein. Methods: The encoding region of Os CSN5 B was amplified by RT-PCR from Nipponbare, a japonica ricecultivar, and ligated into p EASYTM-T5 Zero cloning vector. The insertion was then sub-cloned into the prokary-otic expression plasmid p ET32a+and transformed into Escherichia coli BL21 plys S for inducible expression. The re-combinant CSN5 B fusion protein was purified with Ni-NTA His.Bind Resin and then used for antiserum prepara-tion by immunizing rabbits. The resultant antiserum was used for Western blotting assay. Results Conclusion:The antiserum could react specifically with CSN5 B fusion protein and native CSN5 B protein in the rice plants byWestern blotting. These results laid a foundation for further study on the function of Os CSN5 B.
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