突变型APP双荧光融合基因的构建和FRET对其表达的研究  

Construction of a recombinant plasmid expressing mutant amyloid precursor protein with two types of fluorescence protein and study of its expression by FRET

作　　者：李晓晴[1] 张苏明[1] 骆清铭[2] 张智红[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]华中科技大学生物医学光子学教育部重点实验室,湖北武汉430030

出　　处：《中风与神经疾病杂志》2007年第4期388-390,513,共4页Journal of Apoplexy and Nervous Diseases

基　　金：教育部科学技术研究重大项目(No10420)

摘　　要：目的探讨Alzheimer病的发病机制,研究淀粉样蛋白前体(APP)酶解过程,构建含有Swedish突变的双荧光真核表达系统。方法通过聚合酶链式反应(PCR)得到蓝色荧光蛋白(CFP)和黄色荧光蛋白碱基序列(YFP),生物合成含有Swedish突变的APP中间54个碱基片段(54bp),利用基因工程技术将CFP、54bp、YFP片段克隆至载体质粒pcDNA3.0中,得到重组质粒pcDNA3.0-CFP-54bp-YFP,将其转染至人神经母细胞瘤细胞(SH-SY5Y)中,利用激光共聚焦显微镜观察荧光表达,检测荧光共振能量转移(FRET)。结果酶切和基因序列分析证明重组质粒构建成功;对转染细胞的荧光和FRET检测发现融合基因能够准确表达荧光,同时可以观测到FRET现象。结论含有Swedish突变的荧光融合基因的构建为探索AD的发病机制、活体观察APP裂解奠定了基础。Objective To investigatc the pathogenesis of Alzheimer disease and study the enzymatic progress of amyloid precursor protein,thc recombinant eukaryotic expression plasmid was constructed expressing Swedish mutation and two different types of fluorescent protein. Methods The cyan and yellow fluorescence sequences （which werc named as CFP and YFP respectively） were obtained by polymerase chain reaction. The 54 bases in the middlc of amyloid precursor protcin werc synthesized （which was named as 54bp containing Swedish mutation）. The threc fragments mentioned above （which were CFP,YFP as well as 54bp） were inserted into the vector pcDNA3.0. By genetic engineering the recombinant plasmid peDNA3.0-CFP-54bp-YFP was constructed and finally identified by enzyme digestion and sequencing. Then the construct was transfeeted into SH-SY5Y cells. Its expression was examined by fluorescence confoeal microscopy and the fluorescence resonance energy transfer（FRET） signal was collected. Results DNA sequencing and enzymatic cleavage showed the sequence of the construct was correct. FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. Conclusion It is important to construct such a recombinant plasmid for investigating the pathogenesis of Alzheimer disease and in vivo clarifying the proteolytie processing of amyloid precursor protein.

关 键 词：荧光共振能量转移 淀粉样前体蛋白 融合基因 阿尔茨海默病 

分 类 号：R749.16[医药卫生—神经病学与精神病学]