三种淀粉样前体蛋白胞外结构域突变体的建立及功能验证

The Role of Extracellular Domain of Amyloid Precursor Protein on Amyloid Precursor Protein Expression and Secretion

目的:构建3种淀粉样前体蛋白(APP)胞外结构域突变体细胞株并进行功能的初步验证。方法:通过Touch down PCR的方法扩增E1结构域缺失的APP695片段以及E1和信号肽双缺失的片段,采用融合PCR的方法扩增E2结构域缺失的APP695片段,并将获得的片段连接于pCMV-APP695载体中得到APP胞外结构域突变体质粒;质粒转染CHO细胞后,添加G418筛选表达APP胞外结构域突变体的单克隆细胞系;通过Western blot方法检测APP胞外结构域突变体的表达及胞外分泌水平,分析信号肽对该突变体分泌的影响。结果:成功获得了APP的E1结构域突变体、E1/信号肽双缺失突变体以及E2结构域突变体细胞株,且APP突变体可以正常表达和分泌,但是信号肽缺失后APP突变体可以表达、而不能分泌。结论:APP的E1和E2结构域对APP蛋白的胞外分泌无影响。

Objective: To generate cells that express amyloid precursor protein (APP) with different extracellular domain (ED) deletion or signal peptide (SP),and evaluate role of ED and SP on APP expression and secretion. Methods: APP695 fragments with ED1 deletion and APP fragments with both ED1 and signal peptide (SP) deletion were amplified using touchdown PCR.The APP fragments with ED2 deletion were obtained with fusion PCR.These fragments were further ligated to pCMV-APP695 expression vector to get recombinant expression constructs,which were separately transfected into CHO cells and screened with G418 to get single clone. The effects of ED and SP on expression and secretion of APP were assessed using Western blot. Results: Three cell lines that express APP with ED1 deletion,or ED2 deletion ,or ED1&SP deletion respectively were established successfully.The mutants of APP with SP could express and secret APP fragments in the cells,while the APP mutant without SP could only express APP fragments in the cell but cannot be extracellularly secreted. Conclusions: Both ED1 and ED2 of APP do not influence the secretion of APP.