香草素能瞬变受体亚型-1表达细胞系的构建与鉴定

目的构建香草素能瞬变受体亚型-1(TRPV1)膜蛋白稳定表达的细胞系。用于咳嗽与TRPV1关系的研究,以及新型镇咳药物的开发。方法应用CaCl2法制备大肠杆菌感受态细胞,热激转化,将pCDNA3-TRPV1质粒转入到大肠杆菌感受态细胞中扩增,通过聚乙二醇沉淀法纯化质粒,纯化的pCDNA3-TRPV1质粒用磷酸钙转染法转入HEK293细胞。荧光显微镜观察黄绿色荧光蛋白表达;用5μmol/L的CAP处理转染细胞7h,台盼蓝双染色计算细胞病死率,作为细胞表达的证据。结果经转化并培养后质粒大量扩增,酶切后获得目的基因片段大小约2650bp,大小符合目的基因。转染TRPV1的HEK293细胞在荧光显微镜下可清晰观察到黄绿色荧光。使用5μmol/L的辣椒素处理转染后细胞系7h,发现细胞病死率显著上升。结论 TRPV1在HEK293细胞中稳定表达,可用于构建"咳嗽细胞模型"。

Construction and Identification of Transient Receptor Potential Vanilloid 1 Expression Cell Lines

Objective To construct the stable expression cell lines for transient receptor potential vanilloid-1(TRPV1),in order to study the relationship between cough and TRPV1 and to mark the development of new types of antitussive medicine.Methods Calcium chloride method was used to prepare competent cells of E.coli.The pCDNA3-TRPV1 plasmid was transformed into the E.coli cells by heat shock transformation and then amplified.Plasmid was then purified by polyethylene glycol precipitation,and the pCDNA3-TRPV1 plasmid...