摘要
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 the survival motor neuron （SMN） polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy （SMA）. Methods A prokaryotic expressional plasmid named pET-28a （ ＋ ）/SMN was constructed and SMN-His fusion protein was induced. The fusion protein was used to immunize New Zealand rabbits to prepare SMN polyclonal antibody. A eukaryotic expressional plasmid named pcDNA3. 1/myc-HisB-SMN was constructed and used to transfect CHO cells. Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ , 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis. Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients. Results Correct pET-28a （ ＋ ）/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a（ ＋ ）/SMN. Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein. SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus. The results of Western-blotting were as follows： the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase （GAPDH） band density （SMN/GAPDH） is 0. 619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type III and I1 were 0. 347 and 0. 340 respectively, which were lower than that of normal controls. However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0. 079, which was quite lower than that of normal controls. Conclusions The
出处
《中华神经科杂志》
CAS
CSCD
北大核心
2007年第12期846-849,共4页
Chinese Journal of Neurology
基金
国家自然科学基金(30670730)
福建省自然科学基金计划资助项目(C0610008)
福建省卫生厅青年科研课题基金(2005-1-4)
福建省重大科技项目基金(2002Y001)
