摘要
从水生植物凤眼莲叶片中提取总RNA,经RT-PCR扩增出Ca2+-ATPase基因片段,经限制性内切酶(Sma I,Not I)酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCR和质粒双酶切鉴定、序列测定确认,证实成功地构建了Ca2+-ATPase基因融合表达载体,.转化菌经IPTG诱导表达,获得了大小约48kD的可溶性目的蛋白,与预期相吻合.利用谷胱甘肽琼脂糖凝胶4B(Glutathione Sepharose 4B)亲和介质对重组蛋白进行纯化,获得了高纯度的目的蛋白.
The Ca2＋-ATPase gene was cloned from Eichhornia crassipes leaves using the PCR technol-ogy.After digested by the enzymes （Sma I,Not I）,it was inserted into the plasmid pGEX-4T-2 to re-construct the expression vector.The recombinant protein was induced by IPTG and then purified u-sing Glutathione Sepharose 4B.As a result,a single 48 kDa protein was acquired,which implied the protein was the purified Ca2＋-ATPase fusion protein.
出处
《泉州师范学院学报》
2014年第6期21-24,共4页
Journal of Quanzhou Normal University
基金
国家自然科学基金面上项目(30770391)
关键词
凤眼莲
原核表达
蛋白纯化
Ca2＋-ATPase
Eichhornia crassipes
Ca2＋-ATPase
prokaryotic expression
protein purification
