人肾尿酸转运蛋白1基因克隆、抗体制备及其在肾小管上皮细胞中的定位

目的 制备人尿酸转运蛋白1(hURAT1)多克隆抗体并利用该抗体检测hURAT1在肾组织中的表达及亚细胞定位。方法 用RT-PCR方法从人肾组织中扩增出hURAT1基因全长及其抗原表位区，分别构建绿色荧光蛋白hURAT1-pEGFP融合表达载体和GST融合表达载体pGEX-5X-1。诱导表达并纯化GST融合蛋白，利用改良的蛋白免疫法制备多克隆抗体；Western blot及免疫组化方法观察人肾组织的hUART1基因产物的表达。将hUPAT1-pEGFP重组质粒转染细胞，激光共聚焦显微镜动态观察hURAT1-pEGFP在肾小管上皮细胞LLC-PK1细胞膜的定位及表达。结果 成功获得高效特异的hURAT1抗体，利用该抗体证实hURAT1基因编码产物分布于人肾组织近端肾小管刷状缘，Western blot证实hURAIT1蛋白在肾组织中表达。激光共聚焦显微镜动态观察显示hURAT1-pEGFP定位于LLC-PK1细胞膜。结论 制备的hURAT1抗体及其全长基因可用于hURAT1基因的生理功能及病理研究，hUPAT1基因编码产物分布于人肾组织近端肾小管刷状缘。

Location of hUART1 in proximal tubular epithelial cell by cloning the hUART1 gene and preparing the polyclonal antibody

Objective To prepare the anti-hUART1 polyclonal antibody and investigate the subcellular localization of hURAT1 protein. Methods The full-length hURAT1 gene was obtained by RT-PCR and inserted into the fusion expression vector pEGFP-N3,the predicated antigen epitope was cloned into GST fusion protein expression plasmid pGEX-5X-1, transformed into E. coli BL21 cells for expressing recombinant GST-hURAT1 protein induced by IPTG. The purified hURAT1-GST fusion protein was employed to immunize rabbit for preparing the polyclonal antibody. The expression of hURAT1 was analyzed by western-blot and immunohistochemistry in human kidney. pEGFP-hURAT1 was transfected into the LLC-PK1 cell in order to observe the subcellular localization of the gene using confocal microscopy. Results Specific anti-hURAT1 rabbit polyclonal antibody was obtained, and both Western-blot and immunohistochemistry showed that hURAT1 was expressed in the human kidney brush border,localized in the apical membrane of the LLC-PK1 cell. Conclusions hURAT1 protein was a membrane protein located in renal proximal tubule, which could be detected in the apical membrane. The anti-hURAT1 polyclonal antibody could be used for studying the physiological function of hURAT1 and its pathology.