摘要
将水稻目地基因Rf6连接到载体PMD-18T上,测序正确后将目的片段连接到含有GST标签的p GEX-6P-1原核表达载体上,确认正确的重组质粒转化到BL21菌株;通过LB培养至对数生长期后,加入IPTG进行诱导表达,经SDS-PAGE和Western Blot检测是否诱导出目的条带,并且经过Glutathione Resin亲和层析系统纯化及检测。结果表明,20℃,4 h,0.3 mmol/L IPTG条件下可诱导出可溶性蛋白,经纯化得到可溶性带GST标签的融合蛋白。
To achieve activated fertility restorarion protein in vitro, the gene of rice was cloned into PMD-18 T for sequencing.Then the gene was connected with prokaryotic expression vector p GEX-6P-1 containing the GST-tag and the recombinant prokaryotic expression vector was transformed into E.coli strain BL21. The GST-tagged fusion protein was induced with IPTG in E.coli strain BL21,and confirmed by SDS-PAGE and Western Blot analysis. The results showed that of the soluble fusion protein was induced by 0.3 mmol / L IPTG at 20 ℃ for 4 h, was and purified by Glutathione resin.
出处
《湖北农业科学》
2015年第23期6047-6051,共5页
Hubei Agricultural Sciences
基金
国家自然科学基金项目(31170296)
