中国医科大学学报2017，Vol.46Issue(10)：865-868,4.DOI:10.12007/j.issn.0258-4646.2017.10.001
Cav1.2钙通道C末端远端片段dDCT重组质粒的构建及蛋白制备
Construction of and Protein Preparation from a Recombinant Plasmid Containing the Distal Fragment of the Distal C-terminus of the Cav1.2 Channel
摘要
Abstract
Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.关键词
Cav1.2钙通道/C末端远端片段/pull-down方法Key words
Cav1.2 Channel/distal fragment of the distal C-terminus/pull-down assay分类
医药卫生引用本文复制引用
胡慧媛,郝丽英,雷帅,孙德日,孙旋旋,晏珊,刘世浩,王健,王莹,李越..Cav1.2钙通道C末端远端片段dDCT重组质粒的构建及蛋白制备[J].中国医科大学学报,2017,46(10):865-868,4.基金项目
国家自然科学基金(81100108,31471091) （81100108,31471091）
医学电生理学教育部重点实验室开放基金(201605) （201605）
辽宁省大学生创新创业训练项目(201410159018) （201410159018）
