重症肌无力患者肌细胞中LRP4胞内区与SNX17的相互作用

发布时间：2018-05-02 14:34

本文选题：低密度脂蛋白受体相关蛋白 + 分拣微管连接蛋白　；参考：《郑州大学学报(医学版)》2017年02期

【摘要】：目的:探讨低密度脂蛋白受体相关蛋白4(LRP4)和分拣微管连接蛋白17(SNX17)在重症肌无力(MG)患者肌细胞中相互作用关系。方法:分别构建原核表达载体p ET30a-LRP4胞内区和真核表达载体p EASY-Blunt M2-SNX17,在大肠杆菌BL21(DE3)中诱导表达相对分子质量约为17 800的His-LRP4胞内区,在HEK293T细胞中诱导表达相对分子质量约为53 000的Myc-SNX17。利用抗Myc单克隆抗体磁珠与抗His单克隆抗体磁珠进行双向免疫共沉淀(Co-IP),以验证LRP4、SNX17两种蛋白分子是否存在相互作用。结果:成功构建了原核表达载体pE T30a-LRP4胞内区和真核表达载体pE ASY-Blunt M2-SNX17;抗Myc单克隆抗体磁珠与His-LRP4混合后不发生共沉淀,如再与Myc-SNX17混合则可发生共沉淀;抗His单克隆抗体磁珠与Myc-SNX17混合后不发生共沉淀,再与His-LRP4胞内区混合可发生共沉淀。结论:原核表达的LRP4胞内区蛋白与真核表达的SNX17体外可发生直接结合。
[Abstract]:Aim: to investigate the interaction between low density lipoprotein receptor associated protein (LRP4) and sorting microtubule junction protein 17 (SNX17) in myocytes of patients with myasthenia gravis (MG). Methods: prokaryotic expression vector p ET30a-LRP4 intracellular region and eukaryotic expression vector p EASY-Blunt M2-SNX17 were constructed, respectively. The His-LRP4 region with relative molecular weight of 17 800 was induced in Escherichia coli BL21 DE3. Myc-SNX17 was induced to express about 53 000 relative molecular weight in HEK293T cells. Two-way co-immunoprecipitation was carried out by using Myc monoclonal antibody beads and anti- monoclonal antibody magnetic beads in order to verify the existence of interaction between LRP4 and SNX17 protein molecules. Results: the prokaryotic expression vector pE T30a-LRP4 intracellular region and eukaryotic expression vector pE ASY-Blunt M2-SNX17 were successfully constructed, and the anti-Myc monoclonal antibody magnetic beads did not co-precipitate with His-LRP4, but if mixed with Myc-SNX17, coprecipitation occurred. There was no coprecipitation in the mixture of His monoclonal antibody beads and Myc-SNX17, but coprecipitation occurred in the intracellular region of His-LRP4. Conclusion: prokaryotic expression of LRP4 protein and eukaryotic expression of SNX17 can directly bind in vitro.
【作者单位】：郑州大学第二附属医院神经内科;郑州大学医药科学研究院神经免疫学研究室;郑州大学基础医学院免疫学系;郑州大学第二附属医院普胸外科;河南中医药大学康复医学院康复办公室;
【基金】：国家自然科学基金资助项目81471545 河南省高等学校重点科研项目计划16A320010;17A320046 河南省科技攻关计划项目1721023010298 河南省高校科技创新团队项目16IRTSTHN022
【分类号】：R746.1

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