Na_(V)1.5钠通道C末端IQ基序的重组质粒构建及蛋白制备  

Recombinant plasmid construction and protein expression and purification of the C terminal IQ motif of the Na_(V)1.5 sodium channel

作　　者：张峰慧 薛迎春 许兴荣 邵冬雪 张晨阳 刘岩 苏敬阳 胡慧媛 郝丽英 ZHANG Fenghui;XUE Yingchun;XU Xingrong;SHAO Dongxue;ZHANG Chenyang;LIU Yan;SU Jingyang;HU Huiyuan;HAO Liying(Department of Pharmaceutical Toxicology,School of Pharmacy,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学药学院药物毒理学教研室,沈阳110122

出　　处：《中国医科大学学报》2021年第8期694-698,共5页Journal of China Medical University

基　　金：国家自然科学基金青年基金(81100108);辽宁省教育厅科学研究项目-基础研究项目(JC2019035);辽宁省中央引领地方科研发展专项(2020JH6/105);中国医科大学新冠肺炎疫情防控相关科研攻关项目(2020-07)。

摘　　要：目的构建Na_(V)1.5钠通道C末端IQ基序原核表达载体并进行蛋白表达、纯化和生物学鉴定。方法将IQ基序的cDNA片段插入pGEX-6P-3质粒载体后转化大肠杆菌BL21感受态细胞,异丙基硫代-β-D-半乳糖苷(IPTG)诱导蛋白表达,Glutathione-Sepharos 4B分离纯化后采用SDS-PAGE检测目的蛋白纯度和相对分子质量,pull-down方法检测GST-IQ融合蛋白的生物学活性。结果构建的IQ重组质粒经限制性内切酶和基因测序双重鉴定显示质粒构建成功,融合蛋白经纯化后其浓度和纯度均较高,并具有能够与CSL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可以表达生物活性蛋白的NaV1.5钠通道IQ基序原核表达载体,为深入探讨NaV1.5钠通道的生物学功能奠定了重要的物质基础。Objective To construct an expression plasmid encoding the C terminal IQ motif of the Na_(V)1.5 sodium channel as a glu-tathione-S-transferase(GST)fusion protein,and to express,purify,and characterize the activity of the recombinant protein.Methods A cDNA encoding the NaV1.5 sodium channel IQ motif was ligated into pGEX-6p-3.The resulting plasmid was transformed into Escherichia coli BL21 component cells.Bacteria were cultured to log phase,then induced with IPTG before harvesting.The IQ motif was expressed as a glutathione-S-transferase(GST)fusion protein,which was purified using Glutathione Sepharose 4B beads.SDS-PAGE was used to assess its relative molecular weight and purity.A pull-down assay was performed to investigate the GST-IQ fusion protein’s biological ac-tivity.Results The constructed IQ recombinant plasmid was successfully identified by restriction endonuclease digests and sequencing.Expression and affinity purification resulted in high concentration,high purity target protein that displayed biological activity in the form of concentration-dependent binding to the CSL protein.Conclusion A prokaryotic expression system for expression of the NaV1.5 sodium channel IQ motif has been successfully established,providing an important resource allowing further exploration of the biological function of the NaV1.5 sodium channel.

关 键 词：NaV1.5钠通道 IQ基序 重组质粒 pull down实验 

分 类 号：R966[医药卫生—药理学]