| In-Fusion技术构建Ⅰ型钠通道与红色荧光蛋白共表达载体及其突变载体 |
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| 引用本文： | 刘超,汤斌,曾杨,于美娟,石奕武,廖卫平. In-Fusion技术构建Ⅰ型钠通道与红色荧光蛋白共表达载体及其突变载体[J]. 实用医学杂志, 2011, 27(11): 1908-1910. DOI: 10.3969/j.issn.1006-5725.2011.11.002 |
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| 作者姓名： | 刘超 汤斌 曾杨 于美娟 石奕武 廖卫平 |
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| 作者单位： | 广州医学院第二附属医院神经科学研究所,510260 |
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| 基金项目： | 国家自然科学基金资助项目，广东省自然科学基金资助项目 |
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| 摘 要： | 目的:构建Ⅰ型钠通道(NaV1.1)与红色荧光蛋白(DsRed)共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DsRed真核细胞表达载体(pCMV-IRES-DsRed)。限制性内切酶对载体进行酶切并回收线性载体片段,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-SCN1A-IRES-DsRed。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:成功构建NaV1.1与DsRed共表达载体,SCN1A基因所编码的第1623位密码子由谷氨酸(Glu)突变为丙氨酸(Ala)。结论:NaV1.1与DsRed共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。
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| 关 键 词： | 钠通道 红色荧光蛋白 In-Fusion技术 诱变 SCN1A 癫痫 |
Construction of co-expression vectors of DsRed and wild type NaV1.1 or mutated NaV1.1 using In-Fusion technology |
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| LIU Chao,TANG Bin,ZENG Yang,YU Mei-juan,SHI Yi-wu,LIAO Wei-ping. Construction of co-expression vectors of DsRed and wild type NaV1.1 or mutated NaV1.1 using In-Fusion technology[J]. The Journal of Practical Medicine, 2011, 27(11): 1908-1910. DOI: 10.3969/j.issn.1006-5725.2011.11.002 |
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| Authors: | LIU Chao TANG Bin ZENG Yang YU Mei-juan SHI Yi-wu LIAO Wei-ping |
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| Affiliation: | LIU Chao,TANG Bin,ZENG Yang,YU Mei-juan,SHI Yi-wu,LIAO Wei-ping.Institute of Neuroscience,the Second Affiliated Hospital of Guangzhou Medical College,Guangzhou 510260,China |
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| Abstract: | Objective To construct the co-expression vector of NaV1.1 and DsRed,and the corresponding vector containg mutated NaV1.1.Methods pCMV-IRES-DsRed vector was digested by restriction enzymes and the lined vector was purified.SCN1A gene was performed from pCMV-SCN1A plasmid.SCN1A gene was cloned into the linearized pCMV-SCN1A-IRES-DsRed vector using In-Fusion technology.The corresponding mutational vector was prepared using site-directed mutagenesis kit.The recombinant plasmid was transformed into HEK293T cells... |
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| Keywords: | SCN1A |
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