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

Construction of co-expression vectors of DsRed and wild type NaV1.1 or mutated NaV1.1 using In-Fusion technology

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。

Objective To construct the co-expression vector of Nay1.1 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 using lipofectamine 2000. Nav1.1 expression in HEK293T cells was detected by western blotting assay. Results Co- expression vectors of DsRed and Nav1.1 or mutated Navl.1 were successfully constructed and the mutation site in SCN1A was EI623A. Conclusions The constructed co-expression vector of Nav1.1 and DsRed and its mutational plasmid containing SCNIA EI623A mutation may lay a good foundation for SCN1A function study.