张鹏;崔冬冰;舒莉萍;范安然;
ZHANG Peng;CUI Dongbing;SHU Liping;FAN Anran;National & Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Center for Tissue Engineering and Stem Cell Research, Guizhou Province Key Laboratory of Regenerative Medicine, Guizhou Medical University;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences;

• 1:贵州医科大学细胞工程生物医药技术国家地方联合工程实验室组织工程与干细胞实验中心贵州省再生医学重点实验室
• 2:中国医学科学院成体干细胞转化研究重点实验室

摘要(Abstract)：

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.

关键词(KeyWords)： 淀粉样前体蛋白;胞外结构域;突变体;信号肽;神经退行性疾病
amyloid precursor protein;extracellular domain;mutants;signal peptide;neurodegenerative disease

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技合作计划项目[黔科合LH字(2015)7337];; 国家自然科学基金项目(31660316);; 贵阳市人民政府-贵州医科大学科学技术联合基金(GY2017-8)

作者(Authors): 张鹏;崔冬冰;舒莉萍;范安然;
ZHANG Peng;CUI Dongbing;SHU Liping;FAN Anran;National & Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Center for Tissue Engineering and Stem Cell Research, Guizhou Province Key Laboratory of Regenerative Medicine, Guizhou Medical University;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences;

DOI: 10.19367/j.cnki.1000-2707.2019.06.002

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