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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。
Objective: To investigate the relationship between LRP4 and SNX17 in muscle cells of patients with myasthenia gravis (MG). Methods: The intracellular domain of p ET30a-LRP4 and pEASY-Blunt M2-SNX17 were constructed respectively and induced in E. coli BL21 (DE3) to express intracellularly His-LRP4 with relative molecular mass of 17 800 Region, Myc-SNX17 expressing a relative molecular mass of about 53,000 was induced in HEK293T cells. Two-way co-immunoprecipitation (Co-IP) was performed with anti-Myc monoclonal antibody beads and anti-His monoclonal antibody beads to verify the interaction between the two protein molecules of LRP4 and SNX17. Results: The prokaryotic expression vector pE T30a-LRP4 was successfully constructed and the eukaryotic expression vector pE ASY-Blunt M2-SNX17 was successfully constructed. The anti-Myc monoclonal antibody beads did not co-precipitate with His-LRP4. -SXX17 mixed coprecipitation can occur; anti-His monoclonal antibody beads and Myc-SNX17 mixed coprecipitation does not occur, and then mixed with His-LRP4 intracellular precipitation co-precipitation can occur. Conclusion: Prokaryotic expression of LRP4 intracellular domain protein and eukaryotic expression of SNX17 can occur in vitro direct binding.