人淀粉样前体蛋白基因与绿色荧光蛋白融合基因筛选系统的建立  

The Construction of a Screening System of Human Amyloid Precursor Protein and Enhanced Fluorescence Protein Fusion Gene

作　　者：邱昕[1] 潘纪安[2] 陈宇[2] 张苏明[1] 郭德银[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,湖北武汉430030 [2]武汉大学生命科学院病毒学国家重点实验室,湖北武汉430072

出　　处：《中国神经免疫学和神经病学杂志》2005年第5期287-291,共5页Chinese Journal of Neuroimmunology and Neurology

摘　　要：目的构建携带人淀粉样前体蛋白(APP)的增强型绿色荧光蛋白(EGFP)载体,以利于初步筛选对APPmRNA水平作用的药物。方法将pEGFP质粒和携带野生型APP695亚型的pXCJL经HindⅢ酶切,连接,PCR初筛,酶切鉴定,进一步经测序证实。携带野生型人类APP751亚型cDNA的pCDNA3.1质粒用XbaⅠ酶切,Klenow酶填平,再用HindⅢ酶切,回收APP751片段;质粒EGFP用SmaⅠ和HindⅢ酶切,将目的基因APP751片段与EGFP载体片段连接生成重组质粒,酶切鉴定,测序证实。构建好的重组质粒转染COS-7细胞,观察EGFP的表达情况。结果APP695和APP751重组质粒上的EGFP在COS-7细胞内得以表达。结论构建的APP-EGFP融合基因重组质粒,有助于方便、快速地初筛出作用于APP mRNA水平的药物。Objective In order to screen for medicine that will act on human amyloid precursor protein （APP） at mRNA level, an enhanced fluorescence protein （EGFP） vector haboring human APP was constructed. Methods Both plasmids included pEGFP and pXCJL carring subtype APP695 digested at same a HinⅢ site to generate EGFP vector and APP695 fragment, two fragments were ligated. The possible recombinant plasmids were tested by PCR. The right orientation was identified by restriction endonuclease analysis. The recombinant plasmid was verified by sequencing, pCDNA3. 1 carring APP751 was digested at Xba Ⅰ ,blunted with Klenow fragment. Digested by HindⅢ , APP751 was recovered and ligated with pEGFP vector digested at Sma Ⅰ and Hindm. The recombinant was confirmed by restriction endonuclease analysis and sequencing. COS-7 cells were transfected by recombinant plasmids to test the EGFP expression. Results EGFP carried by two recombinant plasrnids could normally be expressed in COS-7 cells. Conclusions An APP gene screening system was constructed. It is quick and convenient to screen for medicine targeted APP gene at mRNA level.

关 键 词：淀粉样前体蛋白 增强型绿色荧光蛋白 ALZHEIMER病 

分 类 号：R749.16[医药卫生—神经病学与精神病学]