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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 a fusion expression vector of Ⅰ type sodium channel (Nav1.1) and green fluorescent protein (GFP) and its mutated vector. METHODS: The SCN1A gene was subcloned into the pAC GFP1-C In-Fusion Ready Linear Vector using In-Fusion technology. PCMV-GFP-C-SCN1A was obtained by PCR amplification of the SCN1A gene (15 identical bases to both ends of the linear vector) and In-Fusion technology. The cells were transfected into HEK293T cells and the expression of Nav1.1 was detected by Western blot. Site-directed mutagenesis kit for site-directed mutagenesis. The results showed that: 1. The fusion expression vector pCMV-GFP-C-SCN1A was constructed successfully; 2. The DNA sequencing showed that the mutated site at the predicted site, the tryptophan codon 190 (TGG) Mutated to a stop codon (TGA). CONCLUSION: The construction of the fusion expression vector of Nav1.1 and GFP and its mutated vector is successful, which lays the foundation for further study on the function of Nav1.1 induced by this mutation.