摘要： 目的 制备并纯化心肌Na_V1.5通道IQ蛋白，探讨钙调蛋白（CaM）突变体CaM_D130G与IQ基序的结合作用，为后续体外实验明确长QT综合征的发病机制奠定基础。方法 采用同源建模和蛋白对接的方法预测突变体CaM_D130G与IQ蛋白的结合，利用基因重组技术将重组质粒pGEX-6P-1/GST-IQ转化至原核表达载体大肠杆菌中，并诱导表达该蛋白，通过超声破碎方法提取蛋白并使用GS-4B孵育纯化IQ蛋白，SDS-PAGE确认蛋白的制备效果，Pull-down assay方法检测CaM_D130G与IQ蛋白的结合作用。结果 蛋白对接结果显示，CaM/CaM_D130G和心肌Na_V1.5通道IQ蛋白建模成功且具有结合的可能性。SDS-PAGE凝胶电泳结果显示，制备的心肌Na_V1.5通道IQ蛋白浓度和纯度均较高，且与CaM_D130G有很好的结合活性。结论 本研究成功制备出浓度和纯度较高的心肌Na_V1.5通道的IQ蛋白，并探讨了CaM_D130G与心肌Na_V1.5通道IQ基序的结合作用。

基于长QT综合征的钙调蛋白突变体CaM_D130G与心肌钠通道Na_V1.5 IQ基序的结合作用

张晓琳, 邵秀丽, 朱荣利, 张瑞佳, 郝丽英, 封瑞

中国医科大学药学院药物毒理学教研室, 沈阳 110122

收稿日期:2020-06-30出版日期:2021-03-30发布日期:2021-03-20
通讯作者:封瑞E-mail:fengrui527@163.com
作者简介:张晓琳(1995-),女,硕士研究生.
基金资助:国家自然科学基金（31400981）；医学电生理学教育部重点实验室开放基金（201505）


关键词: 长QT综合征, 钙调蛋白, 突变体, 心肌Na_V1.5
Abstract: Objective To prepare and purify cardiac Na_V1.5 channel IQ protein and preliminarily explore its binding to CaM_D130G. This study provides a foundation for further in vitro studies to clarify the pathogenesis of long QT syndrome. Methods Homology modeling and protein docking were conducted to predict the binding between the mutant CaM_D130G and IQ proteins. The recombinant plasmid PGEX-6P-1/GST-IQ was transfected into Escherichia coli and to induce protein expression. The protein was extracted via ultrasonication and purified using the GS-4B incubation method. SDS-PAGE was used to assess IQ protein quality. The binding between CaM_D130G and IQ proteins was detected using pull-down assays. Results The protein docking results indicated that CaM/CaM_D130G and Na_V1.5 channel IQ proteins were successfully modeled and predicted to bind to each other. SDS-PAGE results showed that the prepared cardiac Na_V1.5 channel IQ protein had high concentration and purity and could bind to CaM_D130G. Conclusion In the present study,the cardiac Na_V1.5 channel IQ protein is successfully isolated with high concentration and purity and is demonstrated to bind to CaM_D130G.
Key words: long QT syndrome, calmodulin, mutant, cardiac Na_V1.5
PDF全文下载地址:

https://journal.cmu.edu.cn/CN/article/downloadArticleFile.do?attachType=PDF&id=2706