真核表达质粒pcDNA3.1-tau的构建及在HEK293细胞中的稳定表达

背景：阿尔茨海默病患者的痴呆症状严重程度与脑组织中的神经原纤维缠结数量呈正相关,神经原纤维缠结的主要蛋白成分为过度磷酸化的蛋白tau,tau蛋白的病理改变出现在痴呆症状之前并独立于β-淀粉样多肽的异常。目的：构建tau基因的真核表达质粒,建立稳定表达tau的稳转细胞株。方法：采用反转录-聚合酶链反应方法,从反转录反应合成的人成神经瘤细胞（SH-SY5Y）的总cDNA中,扩增出约1.0kb的tau cDNA片段,用BamHⅠ和XhoⅠ双酶切后定向克隆到真核细胞表达载体pcDNA3.1中,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒;用脂质体介导法将质粒转染入培养的人胚肾细胞,并利用G418进行稳定表达tau的稳转细胞株的筛选,免疫印迹和免疫荧光细胞化学方法检测tau基因的表达。结果与结论：人tau cDNA已克隆到真核细胞表达载体pcDNA3.1中;免疫印迹和免疫荧光细胞化学结果显示人tau基因在人胚肾细胞中获得表达,tau蛋白表达的阳性信号主要位于细胞胞质,说明成功构建了pcDNA3.1-tau的真核表达质粒,建立了稳定表达tau的稳转细胞株。

Construction of eukaryotic expression plasmid pcDNA3.1-tau and its stable expression in HEK293 cell line

BACKGROUND：The number of neurofibrillary tangles in the brains of Alzheimer＇s disease patients parallels to the severity of dementia. The major protein subunit of neurofibrillary tangles is abnormally hyperphosphorylated protein tau. Tau pathology appears before the dementia and is independent of β-amyloid peptide abnormality. OBJECTIVE：To construct eukaryotic expression plasmid of pcDNA3.1-tau and to establish the cell line stably expressing tau protein. METHODS： A 1.0-kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells with the RT-PCR method and was cloned into the vector pcDNA3.1. The plasmid was identified by the double digestion with restriction enzymes BamHI and XhoI and DNA sequencing. Cultured HEK293 cells transfected with the pcDNA3.1-tau plasmid by lipofectamine were selected using G418. The expression of the tau gene was detected by Western blot and immunofluorescence cytochemistry. RESULTS AND CONCLUSION：The human tau gene was successfully cloned into eukaryotic expression vector pcDNA3.1. Immunoblotting and immunofluorescence cytochemistry revealed that the HEK293 cells stably transfected with the pcDNA3.1-tau plasmid expressed a high level of the tau protein in the cytoplasm. This is evidenced that the recombinant eukaryotic expression plasmid of pcDNA3.1-tau was constructed successfully and the HEK293 cell line stably expressing tau protein was established.