Question

如何构建pcDNA3.1表达系统

Answer 1

真核表达载体pcDNA3.1-β淀粉样前体蛋白裂解酶的构建及其在COS-7细胞中瞬时表达
【摘 要】 目的 构建β淀粉样前体蛋白裂解酶（β-site amyloid precursor protein cleaving enzyme,BACE）基因的真核表达载体,为修复阿尔茨海默病(Alzheimer's disease,AD)损伤神经元奠定基础.方法 采用RT-PCR方法,从人的成神经细胞瘤的总cDNA中,扩增出1.5kb的BACE cDNA片段,再用BamHI和XhoI双酶切后定向克隆到真核细胞表达载体pcDNA3.1中,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒；以免疫细胞化学法检测BACE基因的表达情况.结果 人BACE基因的cDNA已克隆到真核细胞表达载体pcDNA3.1质粒中；经脂质体转染COS-7细胞后,并由潮霉素进行筛选,可见转染细胞胞浆中和胞膜上有较高量的BACE蛋白表达.结论 成功构建了pcDNA3.1-BACE的真核表达载体,为研究BACE基因在AD发病机制中的作用及抑制BACE,为治疗AD奠定一定的实验基础.
【关键词】 基因克隆 阿尔茨海默病 β淀粉样前体蛋白裂解酶
中图分类号：Q753 R749.16 文献标识码：A
CONSTRUCTION AND IDENTIFICATION OF EUKARYOTIC EXPRESSION PLASMID PCDNA3.BACE AND ITS TRANSIENT EXPRESSION IN COS-7 CELLS/DONG Weijiang,GONG Huilin,FENG Gaifeng,et al.Department of Anatomy and Histology & Embryology,Medical School of Xi'an Jiaotong University,Xi'an Shaanxi,710061,P.R.China
Corresponding author:HU Haitao,E-mail:huht@mail.xjtu.edu.cn
【Abstract】 Objective To generate eukaryotic expression vector of pcDNA3.1-β-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells.Methods A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1.The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger?dideoxy?mediated chain termination.The expression of the BACE gene was detected by immunocytochemistry.Results The results showed that the cDNA fragment included 1.5 kb total coding region.The recombinant eukaryotic cell expression vector of pcDNA3.BACE was constructed successfully,and the sequence of insert was identical to the published sequence.The COS?7 cells transfected with the pcDNA3.BACE plasmid expressed a high level of the BACE protein in the cytoplasm.Conclusion The recombinant plasmid pcDNA3.BACE can provide a very useful tool for the research on the cause of Alzheimer's disease and lay an important foundation for preventing Alzheimer's disease.
【Key words】 Gene cloning Alzheimer's disease β-site amyloid precursor protein cleaving enzyme
Foundation items:National Natural Science Foundation of China（30400515,30300395）; Doctorate Foundation of the Ministry of Education（20030698011）