重症肌无力患者肌细胞中LRP4胞内区与SNX17的相互作用  

Expression of LRP4 intracellular region and its interaction with SNX17 in muscle cells from patients with myasthenia gravis

作　　者：安颖[1] 吕杰[2] 张迎娜[2] 高峰[2] 张婧[2] 方华[2] 李倩如[3] 杜英[3] 张清勇[4] 王金兰[1] 张运克[5] 

机构地区：[1]郑州大学第二附属医院神经内科,郑州450014 [2]郑州大学医药科学研究院神经免疫学研究室,郑州450052 [3]郑州大学基础医学院免疫学系,郑州450001 [4]郑州大学第二附属医院普胸外科,郑州450014 [5]河南中医药大学康复医学院康复办公室,郑州450000

出　　处：《郑州大学学报（医学版）》2017年第2期129-134,共6页Journal of Zhengzhou University(Medical Sciences)

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

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。Aim： To explore the interaction between LRP4 and SNX17 at the neuromuscular junction（ NMJ） in patients with myasthenia gravis（ MG）. Methods： We constructed prokaryotic expression vector p ET30a-LRP4 intracellular region,transformed it into E. coli BL21（ DE3） bacteria and induced expression of His-LRP4 intracellular region with relative molecular weight of about 17 800; we constructed eukaryotic expression vector p EASY-Blunt M2-SNX17,transfected it into HEK293 T cells and expressed Myc-SNX17 with relative molecular weight of about 53 000. Then we used two-way co-immunoprecipitation to verify whether there was interaction between LRP4 and SNX17. Results： We constructed prokaryotic expression vector p ET30a-LRP4 intracellular region and eukaryotic expression vector p EASY-Blunt M2-SNX17 successfully;Anti-Myc-tag m Ab-magnetic beads mixed with His-LRP4 did not occur co-precipitation,while those mixed with Myc-SNX17 showed co-precipitation; Anti-His-tag m Ab-magnetic beads mixed with Myc-SNX17 did not occur co-precipitation,while those mixed with His-LRP4 intracellular region showed co-precipitation. Conclusion： Prokaryotic expression LRP4 intracellular region and eukaryotic expression SNX17 can combine directly in vitro.

关 键 词：低密度脂蛋白受体相关蛋白4 分拣微管连接蛋白17 重症肌无力 免疫共沉淀 

分 类 号：R746.1[医药卫生—神经病学与精神病学]