摘要
目的明确人尿酸转运蛋白(hUAT)在人肾小管上皮细胞(HKC)内的定位表达情况。方法利用DNA重组技术构建hUAT。绿色荧光蛋白的融合基因分别导入人HKC及非洲爪蟾卵细胞。构建hUAT的谷胱甘肽转移酶(GST)融合表达载体并制备抗hUAT的多克隆抗体。利用免疫荧光、Western印迹及激光共聚焦显微镜等技术观察hUAT在人HKC的定位表达。结果成功制备了兔抗hUAT-GST多克隆抗体。利用该抗体及构建的pEGFP-hUAT荧光表达载体进行Western印迹和免疫荧光检测,结果表明hUAT是一种膜蛋白,并且表达于人HKC胞膜上,Northernblot结果也表明人HKC在高尿酸环境中的hUAT表达水平明显上调(P<0.05)。结论hUAT并非典型的膜转运蛋白,可能是以二聚体的形式才能够表达在细胞膜上,它在细胞内的定位表达可能需要特殊的转录调控机制。
Objective To investigate the location and expression of human uric acid transporter （hUAT）within the human renal tubule epithelial cell. Methods The full-length hUAT/ green fluorescent protein （GFP） fusion plasmid was constructed（hUAT-GFP）, then it was transfected into renal tubule epithelium and Xenopus laevis oocytes. Meanwhile, the GST-hUAT fusion recombinant expression plasmid was constructed and the multi-clonal antibodies against GST-hUAT fusion protein were prepared. The localization of hUAT was determined within Xenopus laevis ooeytes and renal tubule epithelium using immunofluorescence, Western Blot, Northern Blot and confoeal microscopy. Results Rabbit multi-clonal antibodies against GST- hUAT fusion protein were prepared successfully. The hUAT was a membrane protein, which mainly expressed on the plasma membrane of human renal tubular epithelium, hUAT mRNA expression level in human renal tubular epithelium was upregulated significantly when incubated with 400 μmol/L uric acid （ P 〈 0.05）. Condusion hUAT is not a typical membrane protein, and may locate in the membrane in a dimmer form, whose location and expression is mediated through a special transcription regulation mechanism.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2005年第9期527-533,共7页
Chinese Journal of Nephrology
基金
国家自然科学基金"创新研究群体"项目(30121005)国家自然科学基金项目(30100062)国家"973"项目(G2000057000)
