摘要
目的:构建由绿色荧光蛋白(GFP)标记的人醛糖还原酶(AR)融合基因真核表达载体并在HEK293细胞中表达。方法:应用DNA重组技术构建AR与GFP的融合基因(AR-GFP),将AR-GFP插入pcDNA3.1/myc-HisA真核表达载体中,通过磷酸钙共沉淀转染HEK293细胞,倒置荧光显微镜评价转染效率,Western blotting技术检测AR基因在转染细胞中的整合及表达,分光光度法(UV法)测定AR表达活性,用公认的AR抑制剂(ARI)反证活性AR的存在。结果:荧光显微镜对转染细胞的观察及Western blotting结果证实AR-GFP融合蛋白在HEK293细胞中表达,UV法测活、ARI抑制试验均未证实其生物学活性。结论:转染的HEK293细胞可成功地表达AR-GFP融合蛋白,但不能成为ARI真核细胞筛选模型。
AIM： To construct a hAR and GFP fusion gene vector and to observe the AR - GFP gene expression in Hek293 ceils. METHODS： A recombined vector pcDNA3.1/myc - HisA -AR - GFP （pH - AG） was constructed by gene engineering technique. The recombined vector was transfected into Hek293 cells using calcium phosphate. RESULTS ： AR - GFP fusion protein was successfully expressed in Hek293 cells without biologic activity, which was confirmed by fluorescence microscopy and Western blotting. CONCLUSION： The Hek293 cells transfected by AR - GFP fusion gene can express its protein successfully. However, it is not a cellular model for ARI screening.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第11期2138-2141,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30472172)
