β淀粉样蛋白_(1-42)质粒的构建及其与钙调蛋白的结合作用  

Construction of Aβ_(1-42)plasmid and its binding to calmodulin

作　　者：齐爽 孙旋旋 王绮萱 贺意婷 李佳睿 苏敬阳 郝丽英 QI Shuang;SUN Xuanxuan;WANG Qixuan;HE Yiting;LI Jiarui;SU Jingyang;HAO Liying(Department of Pharmaceutical Toxicology,School of Pharmacy,China Medical University,Shenyang 110122,China;Department of Clinical Pharmacology,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 [2]中国医科大学药学院临床药理学教研室,沈阳110122

出　　处：《中国医科大学学报》2024年第6期495-500,共6页Journal of China Medical University

基　　金：辽宁省大学生创新训练项目(S202210159031,S20231015944,S202310159018);医学电生理学教育部重点实验室开放基金(KeyME-2019-07)。

摘　　要：目的探讨β淀粉样蛋白_(1-42)(Aβ_(1-42))与钙调蛋白(CaM)的结合作用。方法采用生物信息学方法获取阿尔茨海默病(AD)异常表达的核心基因,预测AD防治的潜在靶蛋白;通过基因重组的方法构建GST-Aβ_(1-42)重组质粒并进行DNA测序;琼脂糖凝胶电泳进行重组质粒的酶切鉴定;提取纯化GST-Aβ_(1-42)融合蛋白,SDS-PAGE凝胶电泳鉴定是否纯化成功;GST pull-down检测质粒表达的GST-Aβ_(1-42)蛋白与CaM的结合作用。结果获取连接度排名前20的核心基因,将CaM作为靶蛋白;质粒的DNA测序结果证明重组质粒构建成功,琼脂糖凝胶电泳结果显示,酶切后的片段与Aβ_(1-42)片段分子量大小理论值符合,进一步证明重组质粒构建成功;SDS-PAGE凝胶电泳结果显示GST-Aβ_(1-42)蛋白能够与CaM结合,并具有浓度依赖性。结论Aβ_(1-42)重组质粒构建成功,且与CaM具有结合作用。Objective To investigate the involvement of calmodulin(CaM)in the pathogenesis of Alzheimer disease(AD)and the mechanism by which CaM binds to amyloid-β(Aβ).Methods The hub genes expressed in AD and predicted to be the target proteins for AD prevention and treatment were obtained using bioinformatics methods.The GST-Aβ_(1-42)recombinant plasmid was constructed through genetic recombination and was then sequenced.The recombinant plasmids were identified using agarose gel electrophoresis,while the extracted and purified GST-Aβ_(1-42)fusion protein was confirmed using SDS-PAGE gel electrophoresis.GST pull-down assay was used to detect the interaction between GST-Aβ_(1-42)protein and CaM,expressed in the plasmid.Results The top 20 hub genes in degree ranking were obtained.The DNA sequencing results of the plasmid proved that the recombinant plasmid was successfully constructed.The agarose gel electrophoresis results indicated that the fragment digested by the enzyme was similar to the molecular weight of the Aβ_(1-42)gene segments,further proving the successful construction of the recombinant plasmid.Binding of GST-Aβ_(1-42)protein to CaM in a concentration dependent manner was revealed through the GST pull down experiment.Conclusion The GST-Aβ_(1-42)recombinant plasmid is successfully constructed and is shown to bind to CaM.

关 键 词：Β淀粉样蛋白 质粒构建 钙调蛋白 结合能力 活性鉴定 

分 类 号：R966[医药卫生—药理学]