亨廷顿舞蹈病的IT15基因片段真核表达载体构建及其在SH-SY5Y细胞中的表达定位  

Construction of the eukaryotic expression vector encoding IT15 gene fragment and its expression in SH-SY5Y cells

作　　者：田均[1] 李盈枝[2] 殷鑫浈[2] 张宝荣[1] 

机构地区：[1]浙江大学医学院附属第二医院干部保健科,杭州310009 [2]浙江大学医学院附属第二医院神经内科,杭州310009

出　　处：《中华神经科杂志》2008年第4期264-268,共5页Chinese Journal of Neurology

基　　金：浙江省自然科学基金资助项目（J20060713）

摘　　要：目的构建亨廷顿舞蹈病的致病基因IT15基因片段的真核表达载体，观察其在SH—SYSY细胞内的表达分布情况。方法以PCR方法扩增出健康人和亨廷顿舞蹈病患者的IT15基因1号外显子片段，应用基因重组技术与绿色荧光蛋白（GFP）基因融合构建以GFP为报告基因的重组真核表达质粒GFP-Htt-19Q与GFP-mHtt-70Q，并经酶切分析和测序证实。利用脂质体瞬时转染人神经母细胞瘤细胞SH-SYSY细胞；Westernblot证实亨廷顿蛋白氨基末端片段在SH—SYSY细胞内表达；荧光显微镜观察正常与变异亨廷顿蛋白氨基末端片段在细胞内的表达定位情况。结果健康人和亨廷顿舞蹈病患者的IT15基因1号外显子的PCR产物分别为170、310bp的特异性片段；重组体GFP-Htt-19Q与GFP-mHtt-70Q经测序分析读码框正确，转染SH-SYSY细胞后发现正常亨廷顿蛋白氨基末端片段弥散分布于胞质内，胞核内少见，而变异亨廷顿蛋白氨基末端片段在核周及核内形成聚集物。结论构建IT15基因片段真核表达载体是研究亨廷顿舞蹈病的发病机制的有效方法之一；变异亨廷顿蛋白氨基末端片段在核周及核内形成聚集物可能是亨廷顿舞蹈病致病的关键。Objective To construct the eukaryotic expression vector encoding IT15 gene and detect its expression in SH-SYSY cells. Methods The exonl of IT15 gene was amplified from genomic DNA isolated from a healthy adult and a Huntington disease patient lymphoblastoid cell lines by PCR. PCR products were subcloned into eukaryotic expression vector pEGFP-C1. The SH-SYSY cells were transiently transfected with the recombinant plasmids and observed under fluorescence microscope. Western blot was used to detect the expression of the fusion protein. Results PCR products had approximately 170 and 310 bp specific segments. The recombinant eukaryotic expression plasmids were confirmed by restriction endonuclease analysis and sequencing. The N-termlnal fragment of normal huntingtin primarily localized in the cytoplasim of SH-SYSY cells, while the N-termlnal fragment of mutant huntingtin aggregated both perinuclearly and intranuclearly. Conclusions The recombinant eukaryotic expression vectors encoding wild type and mutant IT15 gene fragments are useful for studying pathogenesis of Huntington disease. The Nterminal fragment of mutant huntingtin aggregats both perinuclearly and intranuclearly, which could be important for Huntington disease pathophysiology.

关 键 词：杭廷顿病 神经组织蛋白质类 核蛋白质类 重组蛋白质类 遗传载体 

分 类 号：R686[医药卫生—骨科学]