Location of hUART1 in proximal tubular epithelial cell by cloning the hUART1 gene and preparing the polyclonal antibody
- VernacularTitle: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
- Author:
Di WU
;
Ping ZHANG
;
Xiangmei CHEN
- Publication Type:Journal Article
- Keywords:
anion transport proteins;
uric acid;
fluorescent antibody technique;
antibody
- From:
Medical Journal of Chinese People's Liberation Army
1983;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
