摘要
目的构建Cav1.2钙通道C末端远端片段(dDCT)(2 080~2 169)质粒,表达、提取、纯化蛋白并进行生物学活性鉴定。方法将dDCT的cDNA片段插入pGEX-6p-1质粒载体并转化大肠杆菌,异丙基硫代-β-D-半乳糖苷诱导蛋白表达,超声破碎法提取蛋白。GS-4B beads纯化蛋白后,pull-down方法分析其生物学活性。结果构建的dDCT质粒经限制性内切酶和测序双重鉴定成功,经超声破碎法提取的dDCT蛋白纯度和浓度均较高,并具有能够与GST-CT1融合蛋白浓度依赖性结合的生物学活性。结论本研究成功构建了dDCT重组质粒,为深入探讨Cav1.2钙通道的自身调节机制奠定了重要的物质基础。
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 Cav1.2 channel autoregulation.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2017年第10期865-868,共4页
Journal of China Medical University
基金
国家自然科学基金(81100108
31471091)
医学电生理学教育部重点实验室开放基金(201605)
辽宁省大学生创新创业训练项目(201410159018)
