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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 determine the localization of human uric acid transporter (hUAT) in human renal tubular epithelial cells (HKC). Methods Recombinant hUAT was constructed by DNA recombination technology. Fusion genes of green fluorescent protein were introduced into human HKC and Xenopus egg respectively. Construction of hUAT glutathione transferase (GST) fusion expression vector and preparation of anti-hUAT polyclonal antibody. Immunofluorescence, Western blot and confocal microscopy were used to observe the localization of hUAT in human HKC. Results Rabbit anti-hUAT-GST polyclonal antibody was successfully prepared. Western blotting and immunofluorescence analysis using this antibody and the constructed pEGFP-hUAT fluorescent expression vector showed that hUAT was a membrane protein and expressed on the human HKC cell membrane. Northern blot results also indicate that human HKC is involved in the hyperuricemic environment hUAT expression was significantly increased (P <0.05). Conclusion hUAT is not a typical membrane transporter and may be expressed as a dimer on the cell membrane. Its localization in the cell may require special transcriptional regulation.