绿色荧光蛋白与人肾脏NaDC3融合基因的构建及其在肾小管上皮细胞中的定位

本采用RT-PCR方法从人的正常肾组织中扩增出hNaDC3基因全长cDNA序列，采用基因重组技术将hNaDC3基因和绿色荧光蛋白基因融合，通过真核表达质粒—脂质体介导，导入LLC-PKl细胞系，激光共聚焦显微镜动态观察GFP—hNaDC3的定位情况。结果显示PKGFP-C3空白质粒转染的LLC—PKl细胞表达了GFP，GFP在胞内分布以核浆为主，呈均匀致密的细颗粒荧光，胞核染色强，核仁荧光稀少，界线清楚。而pKGFP-C3-hNaDC3转染细胞株的绿色荧光蛋白，转染后第1天混合分布于细胞浆和细胞膜，以粗颗粒荧光为主，两界限不清楚，胞浆中可见未染色的圆形空洞，未见胞核染色；第3天主要聚集于细胞膜，核周的胞浆中可见粗颗粒荧光物质，胞浆和胞膜界线清楚，胞核亦未见绿色荧光蛋白；第5天清晰聚集于细胞膜，细胞膜连接至网状。因此hNaDC3蛋白在细胞质中生成后，定位于细胞膜并稳定表达。

CONSTRUCTION OF RECOMBINANT hNaDC3/GFP AND ITS LOCALIZATION IN RENAL PROXIMAL TUBULE EPITHELIA

To construct the fusion gene of hNaDC3 with green fluorescent protein (GFP), and study the localization of the fusion protein in renal proximal tuble epithelia, the full length cDNA of hNaDC3 was amplified by RT-PCR from normal human renal tissues, and then cloned into pGEM-T vector. After DNA sequencing, the hNaDC3 was inserted into mammalian expression plasmid pEGFP-C3 successfully. The fused gene was then transfected into LLC-PK1 cell with lipofectamine. Confocal fluorescence microscopy showed that green fluorescence spread over the whole cell transfected with pEGFP-C3. After transfection with pEGFP-C3-hNaDC3, fluorescence distributed in both the cytoplasm and on the plasma membrane first day, located mainly on the plasma membrane fifth day. It is suggested that the hNaDC3 protein localizes on the plasma membrane.