津田芜菁MYB77蛋白的原核表达及纯化

目的:建立津田芜菁转录因子MYB77的原核表达系统,并在大肠杆菌中获得表达。方法:RT-PCR获得MYB77的编码序列,将其克隆至pGEM-T载体中,在上下游引物中分别引入HindⅢ和EcoRⅠ酶切位点,PCR获得带酶切位点的目的片段并将其连接到重组表达载体pGEX-KG中,转化大肠杆菌DE3工程菌株,IPTG诱导重组质粒pGEX-KG-MYB77在大肠杆菌DE3中表达带有GST标签的融合蛋白,超声裂解大肠杆菌,用MagneGST ProteinPurification System纯化目的蛋白,通过SDS-PAGE和Western印迹验证GST-MYB77融合蛋白的表达。结果:重组菌株可以表达GST-MYB77融合蛋白,用Western印迹鉴定纯化的融合蛋白,在相对分子质量为55.56×103处检测到目的条带。结论:利用大肠杆菌表达系统获得了较高纯度的GST-MYB77融合蛋白,为进一步研究津田芜菁MYB77蛋白的功能奠定了基础。

Prokaryotic Expression and Purification of the Protein MYB77 of Brassica rapa

Objective： To construct prokaryotic expression vector carrying DNA fragment encoding MYB77 of Bras-sica rapa and to express MYB77 in E.coli.Methods： The DNA fragment encoding MYB77 was obtained by RT-PCR and cloned into pGEM-T.DNA fragment with HindⅢ and EcoRⅠ sites was sequenced correctly and digested to ligate into pGEX-KG.Then the recombination vector pGEX-KG-MYB77 was transformed into E.coli Rosetta（DE3） strain to express fusion protein GST-MYB77 by IPTG induction.Bacterial bodies were disrupted by sonica-tion,and the soluble fraction of fusion proteins were purified by MagneGST protein purification system.GST-MYB77 fusion protein was verified on SDS-PAGE by Coomassie brilliant blue R250 staining and Western blotting analysis.Results： Recombinant expression plasmid pGEX-KG-MYB77 was obtained.The band of purified protein whose molecular weight was about 55.56 kD could be detected by antibody against GST.Conclusion： GST-MYB77 fusion protein was obtained,which will contribute to the study of function of MYB77 in Brassica rapa.