| 刘超 段现来 肖乐 王爱民 欧阳松 曾乐平.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[J].现代生物医学进展英文版,2015,15(21):4037-4039. |
| 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 MutationalVector by In-fusion Technology |
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| DOI： |
| 中文关键词: In-Fusion 技术 诱变 SCN1A 癫痫 |
| 英文关键词: In-Fusion technology Mutagenesis SCN1A Epilepsy |
| 基金项目:国家自然青年科学基金项目（81100663） |
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| 中文摘要: |
| 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（pAcGFP1-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pCMV-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pCMV-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。 |
| 英文摘要: |
| Objective:To construct the fusion protein vector of Nav1.1-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 cells and the expression of Nav1.1 was detected
by western blot. Its mutational vector was performed by commercial site-directed mutagenesis kit.Results:1. The fusion protein
vector of Nav1.1-GFP was successfully constructed. 2. As shown by DNA sequencing, mutation was identified at the directed site as
SCN1A R190X.Conclusion:The successful fusion protein vector of Nav1.1-GFP and its mutational plasmids SCN1A R190X may provide
a molecular basis for further functional and genomic investigation of SCN1A. |
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