In-Fusion技术构建Ⅰ型钠通道与绿色荧光蛋白融合表达及其突变载体  

The Construction of Fusion Protein Vector of Nav1.1-GFP, and its Mutational Vector by In-fusion Technology

作　　者：刘超 段现来 肖乐 王爱民 欧阳松 曾乐平[3] 

机构地区：[1]长沙市第一医院,湖南长沙410005 [2]长沙市第三医院,湖南长沙410015 [3]中南大学湘雅医学院,湖南长沙410013

出　　处：《现代生物医学进展》2015年第21期4037-4039,4055,共4页Progress in Modern Biomedicine

基　　金：国家自然青年科学基金项目(81100663)

摘　　要：目的:构建Ⅰ型钠通道(Nav1.1)与绿色荧光蛋白(GFP)融合表达载体及其突变载体。方法:利用In-Fusion技术将SCN1A基因亚克隆到绿色荧光蛋白真核细胞融合表达载体(p Ac GFP1-C In-Fusion Ready Linear Vector)。PCR扩增SCN1A基因(与线性载体对应两端有15个相同碱基),In-Fusion技术进行融合即得到p CMV-GFP-C-SCN1A。将其转染HEK293T细胞,Western blot检测Nav1.1的表达。定点诱变试剂盒对其进行定点诱变。结果:1.成功构建Nav1.1与GFP融合表达载体p CMV-GFP-C-SCN1A;2.DNA测序表明:在预期位点已经发生突变,SCN1A基因第190位色氨酸密码子(TGG)突变为终止密码子(TGA)。结论:Nav1.1与GFP融合表达载体及其突变载体的构建成功,为进一步研究该突变位点导致Nav1.1功能的改变奠定了基础。Objective： To construct the fusion protein vector of Navl.I-GFP, and its mutational vector. Methods： The In-Fusion technology was applied to subclone SCN1A gene to pAcGFP1-C in-fusion ready linear vector. PCR amplification of SCN1A gene was performed （there are same 15 bases corresponding to the lined vector ends）. In-Fusion technology would be to clone SCN1A gene PCR products into the pAcGFP1-C in-fusion ready linear vector. It was transfected into HEK 293T ceils and the expression ofNavl. 1 was de- teeted by western blot. Its mutational vector was performed by commercial site-directed mutagenesis kit. Results： 1. The fusion protein vector of Navl. 1-GFP was successfully constructed. 2. As shown by DNA sequencing, mutation was identified at the directed site as SCN1A R190X. Conclusions： The successful fusion protein vector of Nav1.1-GFP and its mutational plasmids SCN1A R190X may pro- vide a molecular basis for further functional and genomic investigation of SCN1A.

关 键 词：In-Fusion技术 诱变 SCN1A 癫痫 

分 类 号：Q75[生物学—分子生物学] Q78