微管结合蛋白Tau磷酸化位点突变体的构建及鉴定

Construction and Detection of Tau Phosphorylation Site Mutation

目的:构建带有Myc标签的Thr-213、Thr-235、Ser-404磷酸化位点突变为Ala的Tau蛋白真核表达质粒,并在真核细胞中表达Myc-Tau3m蛋白。方法:利用重组PCR方法得到Thr-213、Thr-235、Ser-404磷酸化位点突变为Ala的编码序列,将其插入经Bam HⅠ和KpnⅠ酶切的p XJ40-Myc表达载体,在人293T细胞中转入空载体和重组质粒,利用Western印迹检测其表达及功能。结果:测序和酶切结果证实Myc-Tau3m真核表达质粒构建成功,转染293T细胞后获得表达,表达产物可促进微管的乙酰化。结论:构建了Thr-213、Thr-235、Ser-404磷酸化位点突变的Myc-Tau3m真核表达载体,为进一步研究Tau蛋白磷酸化的生理机能奠定了基础。

Objective： To construct the human Tau（T231A, S235A, S404A） mutation and detect its function in regulating microtubules acetylation. Methods： The human Tau（T231A, 5235A, S404A） mutation was obtained by recombinant PCR and cloned into pXJ40-Myc vector. The recombinant plasmid was transfected into 293T cells, and the expression of Myc-Tau3m and mierotubules acetylation was detected by Western blot. Results： Myc- Tau3m eukaryotie expression vector labeled with Mye tag was successfully constructed. The inserted fragment was confirmed by sequencing. Myc-Tau3m was expressed in 293T cells and enhanced the acetylation of microtubules. Conclusion： The eukaryotic expression vector of Myc-Tau3m was successfully constructed and expressed in 293T cells.