摘要
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hURAT1pEGFP融合表达载体和GST融合表达载体pGEX5X1。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;Westernblot及免疫组化方法观察人肾组织的hUART1基因产物的表达。将hURAT1pEGFP重组质粒转染细胞,激光共聚焦显微镜动态观察hU RAT1pEGFP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hU RAT1基因编码产物分布于人肾组织近端肾小管刷状缘,Westernblot证实hURAT1蛋白在肾组织中表达。激光共聚焦显微镜动态观察显示hURAT1pEGFP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,hURAT1基因编码产物分布于人肾组织近端肾小管刷状缘。
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.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2005年第5期397-400,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(编号30100062)
国家重点基础研究发展规划(编号G2000057000)
国家自然科学基金青年创新研究群体基金(编号30121005)资助课题
关键词
阴离子转运蛋白质类
尿酸
荧光抗体技术
抗体
anion transport proteins
uric acid
fluorescent antibody technique
antibody
