摘要： 目的 构建Na_V1.5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CS_L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Na_V1.5钠通道IQ基序原核表达载体，为深入探讨Na_V1.5钠通道的生物学功能奠定了重要的物质基础。

Na_V1.5钠通道C末端IQ基序的重组质粒构建及蛋白制备

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

中国医科大学药学院药物毒理学教研室, 沈阳 110122

收稿日期:2020-12-01出版日期:2021-08-30发布日期:2021-07-29
通讯作者:胡慧媛E-mail:hyhu@cmu.edu.cn
作者简介:张峰慧(1997-),女,硕士研究生.
基金资助: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）


关键词: Na_V1.5钠通道, IQ基序, 重组质粒, pull down实验
Abstract: Objective To construct an expression plasmid encoding the C terminal IQ motif of the Na_V1.5 sodium channel as a glutathione-S-transferase (GST) fusion protein,and to express,purify,and characterize the activity of the recombinant protein. Methods A cDNA encoding the Na_V1.5 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 activity. 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 CS_L protein. Conclusion A prokaryotic expression system for expression of the Na_V1.5 sodium channel IQ motif has been successfully established,providing an important resource allowing further exploration of the biological function of the Na_V1.5 sodium channel.
Key words: Na_V1.5 sodium channel, IQ motif, recombinant plasmid, pull down assay
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