INTRACELLULAR TRAFFICKING MECHANISM OF TRANSPORTERS LOCATED ALONG THE CANALICULAR MEMBRANE OF HEPATOCYTES

1.表达载体的构建构建了人P-糖蛋白(人多药耐药基因hMDR1)和绿色荧光蛋白(GFP)的融合蛋白表达载体。其中一株表达GFP-hMDR1，另一株表达hMDR1-GFP。Western blotting将这些载体导入HeLa细胞，细胞裂解液用于Western blotting。用抗GFP和C219(抗P-糖蛋白)检测到220kD的反应，表明这些载体可以在细胞内表达GFP和hMDR1.3的融合蛋白。融合蛋白的定位GFP-hMDR1融合蛋白在不同类型的细胞中广泛分布，结果与单独表达GFP的结果一致。另一方面，hMDR1-GFP沿细胞质膜分布。因此，该载体可表达hMDR1-GFP蛋白，对研究hMDR1-GFP的细胞内转运机制具有重要意义。

1. Construct of expression vectorWe made two types of vectors which expressed a fusion protein of human p-glycoprotein (human multidrug-resistance 1, hMDR1) and green fluorescent protein (GFP). One of them was made to express GFP-hMDR1, the other hMDR1-GFP.2. Western blottingThese vectors were transfected into HeLa cells and the cell lysate was served for Western blotting. We could detect a reaction of 220 kD using anti-GFP and C219 (anti-p-glycoprotein), suggesting that these vectors could intracellularly express fusion proteins of GFP and hMDR1.3. Localization of fusion proteinThe localization of the GFP-hMDR1 fusion protein was diffuse in various types of cells, which result was the same as GFP alone. On the other hand, hMDR1-GFP was found along the plasma membrane. Therefore, this vector, which expresses the hMDR1-GFP protein, would be very useful to investigate intracellular trafficking mechanism.