突变APP荧光融合基因的构建和表达  

Construction and Expression of Recombinant Plasmid Containing APP Flemish Mutation Fused with Fluorescence Sequence

作　　者：李晓晴[1] 薛峥[1] 张苏明[1] 

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,武汉430030

出　　处：《华中科技大学学报（医学版）》2007年第6期795-797,F0004,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：教育部科学技术研究重大项目(No.10420);国家自然科学基金资助项目(No.30700244)

摘　　要：目的探讨阿尔茨海默病(Alzheimer disease)的发病机制,研究淀粉样蛋白前体(amyloid protein precursor,APP)酶解过程,构建含有APP Flemish突变的荧光真核表达系统。方法通过聚合酶链式反应(PCR)得到含有Flem-ish突变的APP最后300 bp片段(C99)、黄色荧光蛋白碱基序列(yellow fluorescence protein,YFP),利用基因工程技术将YFP、C99片段克隆至载体质粒pcDNA3.0中,通过酶切,测序鉴定最终得到重组质粒pcDNA3.0-YFP-C99,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融合基因表达和Aβ的生成,MTT检测转染细胞活性。结果①基因序列分析证明重组质粒构建成功;②激光共聚焦显微镜下观察黄色荧光分布于转染细胞内,表明融合基因能够准确表达荧光;③MTT检测显示Aβ生成后细胞活性下降,差异有极显著性意义(P<0.01);④激光共聚焦显微镜下观察YFP-C99能够生成Aβ,转染细胞内有荧光颗粒聚集沉积,形态异常。结论荧光标记APP Flemish突变重组质粒构建成功,为进一步在体研究APP的有序裂解特别是γ裂解活动奠定了基础。Objective To investigate the pathogenesis of Alzheimer disease and study the enzymolysis of amyloid protein precursor （APP） and construct the recombinant eukaryotic expression plasmid containing APP Flemish mutation and yellow fluorescence sequence. Methods The last 300 bases of APP （which was named as C99 containing Flemish mutation）, together with yellow fluorescence sequence （which was named as YFP） were obtained by polymerase chain reaction （PCR）. The two fragments, YFP and C99, were inserted into the vector pcDNA3.0. By genetic engineering the recombinant plasmid pcDNA- 3.0-YFP-C99 was constructed and finally identified by enzyme digestion and sequencing. Then the construct was transfected in- to SH-SY5Y cells. The expression of fusion gene and generation of Aβ was examined by laser confocal microscopy. The cytotoxicity of Aβ was detected by MTT assay. Results ① DNA sequencing showed the sequence of the construct was correct;② The yellow fluorescence could be seen within the cells by the spectrum confocal fluorescence microscopy, indicating the correct expression of fusion gene;③ The cell viability was decreased after Aβ generation with the difference being significant （P〈 0.01） ;④ Under the confocal microscopy, it was found that YFP-C99 could generate the Aβ, and the transfected cells had the accumulation of fluorescent particles and abnormal morphology. Conclusion The recombinant plasmid containing the APP Flemish mutation labeled by the YFP was successfully constructed, which can make a base to further study the APP sequential cleavage, especially the cleavage by the γ, secretase.

关 键 词：阿尔茨海默病 淀粉样蛋白前体 Flemish突变 基因融合 

分 类 号：R27[医药卫生—中医学]