人胆酸转运蛋白基因的克隆、亚细胞定位及其在肾组织中的表达

目的：克隆人肾与小肠ASBT（顶端Na+/胆汁酸协同转运蛋白）基因并比较2者的序列差别，明确ASBT蛋白在肾小管上皮的亚细胞定位及在人肾组织中的表达情况。方法:从人肾和小肠组织中提取总RNA，然后用带有8肽FLAG标签的PCR引物通过RT-PCR技术扩增ASBT全长cDNA基因并测序，并将其插入真核表达载体中构建ASBT蛋白真核表达载体，然后将其转染到肾小管上皮细胞LLC-PK1中表达并用免疫荧光-激光共聚焦显微镜观察该蛋白的亚细胞定位情况。用免疫组化技术观察ASBT在人肾组织中的表达分布。结果:序列分析结果表明肾小管ASBT基因的序列与小肠ASBT序列完全一致。Western blotting表明ASBT基因在LLC-PK1细胞中得到了正确的表达。共聚焦显微镜分析显示正常ASBT蛋白主要定位于肾小管上皮细胞膜上，与生物信息学的预测结果一致。免疫组化染色表明ASBT蛋白主要表达于人近端肾小管上皮的刷状缘侧，在间质及远端小管没有表达。结论:人肾小管ASBT基因序列与小肠ASBT相同，ASBT蛋白主要表达于近端肾小管上皮细胞管腔侧细胞膜。

Gene cloning, subcellular localization and expression in kidney tissue of human ASBT protein

AIM:To compare the cDNA sequences of ASBT (apical Na+/bile acid cotransporter) gene cloned from human kidney and intestine tissues, and to determine subcellular localization of ASBT in renal tubular epithelial cells and expression site of ASBT in human kidney tissue. METHODS:The total RNA was extracted from human kidney and intestine tissues. The full-length cDNA gene of ASBT was amplified by RT-PCR technique using primers with 8-peptide FLAG tag, sequenced and inserted into eukaryotic expression vector to construct ASBT protein expression vector, which was then transfected into swine renal tubular epithelial cell line LLC-PK1. The subcellular localization of ASBT protein was determined by immunofluorescence staining and laser confocal microscope. Immunohistochemical analysis was used to observe expression position of ASBT in kidney tissue. RESULTS:The results revealed that the sequence of kidney ASBT cDNA gene was identical to that of human intestine ASBT gene. Western blotting analysis indicated that ASBT protein was correctly expressed in LLC-PK1 cells. Confocal scanning analysis showed that ASBT protein was mainly localized at the cellular plasma membrane, consistent with the predicting result obtained by bioinformatics. The results of immunohistochemistry revealed that ASBT was expressed at brush border membrane of proximal renal tubular cells, but not expressed in distal tubule and renal interstitium. CONCLUSION:The gene sequence of kidney ASBT is the same as that of intestine ASBT and ASBT protein is expressed at the lumen (apical) membrane of proximal renal tubular epithelial cells.