部分性发作癫痫相关SCN1A基因M145fsX148突变体的构建及亚细胞定位  

Construction of partial seizure related SCN1A gene M145fsX148 mutation and its subcellular localization

作　　者：罗金杰[1] 李文斌[1] 汤斌[1] 廖卫平[1] 

机构地区：[1]广州医学院第二附属医院神经科学研究所,广东广州510260

出　　处：《中风与神经疾病杂志》2013年第1期4-7,共4页Journal of Apoplexy and Nervous Diseases

摘　　要：目的构建部分性发作癫痫SCN1A基因M145fsX148突变体与黄色荧光蛋白(YFP)融合基因表达载体pCMV-YFP-MuSCN1A,观察其在人类神经母细胞瘤株SH-SY5Y细胞中的亚细胞定位。方法应用基因重组技术,构建YFP与部分性癫痫SCN1A基因M145fsX148融合表达质粒载体,脂质体法转染技术将其与荧光真核表达载体pECFP-ER共转入SH-SY5Y细胞,采用激光扫描共聚焦显微镜观察其亚细胞定位情况。结果重组质粒经酶切鉴定和测序分析证实构建成功,激光扫描共聚焦显微镜显示重组真核表达载体pCMV-YFP-MuSCN1A主要在SH-SY5Y细胞胞质中表达。结论成功构建部分性发作癫痫相关SCN1A基因M145fsX148突变体与YFP融合基因质粒载体,实验显示其在SH-SY5Y细胞胞质中表达,为该突变基因的进一步研究奠定了基础。Objective To construct a plasmid expressing yellow fluorescent protein （YFP） and gene of SCN1 A M145fsX148 mutation gene in partial seizures, and to investigate the localization of this gene in SH-SYSY cell line. Meth- ods An plasmid vector,pCMV-YFP-MuSCN1A containing the gene SCN1A truncation mutation （M145fsX148）and YFP were constructed by techniques of gene recombination and screening, and confirmed by enzyme digestion and gene sequen- cing. The plasmid and pECFP-ER were transfected into SH-SY5Y cell line and their subeellular localizations were detected using confoeal laser scanning microscope. Results Digestion and sequencing analysis showed that the recombinant plasmid vector （pCMV-YFP-MuSCN1A） was correct. The plasmids were successfully transfeeted into SH-SYSY cells. Yellow fleuorescent was found to be localized in the cytoplasm. Conclusion A plasmid vector containing the M145fsX148 muta- tion and YFP has been constructed successfully and its specificly expressedion in the cytoplasm of SH-SY5Y cells. This forms the basement of the further study of mutation.

关 键 词：部分性发作癫痫 SCN1A基因 质粒构建 亚细胞定位 

分 类 号：R742.1[医药卫生—神经病学与精神病学]