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目的制备运动神经元生存(SMN)蛋白多克隆抗体,探讨 SMN 蛋白在细胞内的定位及在脊髓性肌萎缩症(SMA)患者骨骼肌中的表达情况。方法构建 pET-28a(+)/SMN 原核表达质粒,诱导表达 SMN-His 融合蛋白,免疫新西兰大白兔制备 SMN 多克隆抗体。构建 pcDNA3.1/myc-HisB-SMN 真核表达质粒并转染中国仓鼠卵母(CHO)细胞。收集骨折患者以及Ⅰ、Ⅱ、Ⅲ型 SMA 患者的骨骼肌组织各3份。分别采用免疫印迹及免疫荧光染色技术进行 CHO 细胞及骨骼肌组织的 SMN蛋白表达研究。结果成功制备了兔抗人全长 SMN 多克隆抗体,经鉴定其特异性及敏感性均较高。免疫荧光染色显示 SMN 蛋白在细胞质及细胞核中呈斑片状或颗粒状分布,以核周较为明显。免疫印迹结果显示骨折患者骨骼肌组织 SMN 与内参照磷酸甘油醛脱氢酶(GAPDH)条带密度比值(sMN/GAPDH)平均为0.619,Ⅲ、Ⅱ型 SMA 患者 SMN/GAPDH 比值较低,均值分别为0.347和0.540,而Ⅰ型 SMA 患者骨骼肌中 SMN/GAPDH 比值显著降低,均值仅为0.079。结论高质量的兔抗人全长 SMN 多克隆抗体的制备为 SMA 的蛋白功能及发病机制研究奠定了基础,SMA 患者骨骼肌中 SMN 蛋白表达量可能与疾病的严重程度相关。
Objective To prepare polyclonal antibody against motor neuron survival (SMN) protein and to investigate the localization of SMN protein in intracellular and the expression of SMN in skeletal muscle of patients with spinal muscular atrophy (SMA). Methods The prokaryotic expression plasmid pET-28a (+) / SMN was constructed and the SMN-His fusion protein was induced to express the polyclonal antibody against SMN. The pcDNA3.1 / myc-HisB-SMN eukaryotic expression plasmid was constructed and transfected into Chinese hamster ovary (CHO) cells. Three patients with skeletal muscle and three patients with type I, II and III SMA were collected. The expression of SMN protein in CHO cells and skeletal muscle tissues was studied by Western blotting and immunofluorescence staining respectively. Results The rabbit anti-human SMN polyclonal antibody was successfully prepared and its specificity and sensitivity were high. Immunofluorescence staining showed that SMN protein was patchy or granular distribution in the cytoplasm and nucleus, especially in the nuclei. The results of Western blotting showed that the average ratio of sMN / GAPDH was 0.619, the SMN / GAPDH ratios of skeletal muscle tissue were lower, the mean values of SMN / GAPDH were lower 0.347 and 0.540, while the type I SMA patients with skeletal muscle SMN / GAPDH ratio was significantly lower, with an average of only 0.079. Conclusion The preparation of high quality rabbit anti-human SMN polyclonal antibody lays the foundation for the study of protein function and pathogenesis of SMA. The expression of SMN protein in skeletal muscle of SMA patients may be related to the severity of the disease.