携带绿色荧光蛋白基因的APP695真核细胞表达载体的构建及鉴定  

Construction and identification of a eukaryotic expression vector for APP695 gene containing green fluorescence protein gene

作　　者：梁静[1] 李宁[1] 王凤斌[1] 张丽娟[1] 鞠吉雨[2] 

机构地区：[1]潍坊医学院应用药理学实验室,潍坊261042 [2]潍坊医学院免疫学实验室,潍坊261042

出　　处：《中华神经医学杂志》2008年第12期1193-1195,共3页Chinese Journal of Neuromedicine

基　　金：山东省自然科学基金资助课题（Y2004C29）

摘　　要：目的构建大鼠淀粉样前体蛋白（APP）695基因真核细胞荧光表达载体，进行细胞转染、表达，为阿尔茨海默病（AD）治疗药物的研究提供一种有效的细胞模型。方法设计并合成APP695基因的引物．以携带APP695基因的真核细胞表达载体pCB6质粒为模板，PCR扩增得到APP695基因全序列：将APP695基因连接到携带绿色荧光蛋白基因的载体pIRES2-EGFP上；应用酶切分析、PCR检测及DNA测序分析等鉴定所构建的真核细胞表达载体。结果以重组载体为模板扩增得到的片段大小与已知APP695基因大小相同，酶切也得到目的基因片段，测序结果显示与已知APP695基因序列相同。结论成功构建了携带绿色荧光蛋白基因的APP695基因真核细胞表达载体,为AD治疗药物的研究提供了一种有效的分子工具。Objective To construct a eukaryotic expression vector for A PP695 gene carrying green fluorescence protein （GFP） gene. Methods RT-PCR was used to amplify the full-length A PP695 cDNA. The PCR products and the eukaryotic expression vector plRES2-EGFP were digested by restriction endonucleases, and the digested APP695 gene was inserted into the digested eukaryotic expression vector. The positive recombinants were identified by PCR analysis, Nhe Ⅰ and Hind Ⅲ digestion and sequence analysis. Results The 2088-bp DNA fragment was amplified by PCR from the plasmid pCB6, and an identical DNA fragment was also amplified from the recombinants. The products of double restriction enzyme digestion were APP695 gene with a 5.3-kb DNA fragment. Sequence analysis confirmed successful insertion of A PP695 gene into plRES2-EGFP vector. Conclusion The eukaryotic expression vector pIRES2/APP695-EGFP has been successfully constructed.

关 键 词：阿尔茨海默病 淀粉样前体蛋白695 真核表达载体 

分 类 号：R749.16[医药卫生—神经病学与精神病学]