人钠/二羧酸协同转运蛋白1基因融合表达及其抗体制备

利用DNA重组技术 ,将编码人钠 羧酸协同转运蛋白 1(hNaDC1)抗原表位区 (W138 Q2 19)的cDNA克隆至融合表达载体pGEX 5X 1,构建重组质粒pGEX hNaDCL6 .在大肠杆菌BL2 1中 ,经IPTG诱导 ,获得谷胱甘肽巯基转移酶 (GST) hNaDC1重组融合蛋白的表达 .以谷胱甘肽 Sepharose 4B亲和层析 ,获得纯化的GST hNaDC1.以此为免疫原制备的抗hNaDC1抗体可特异性识别人类和大鼠肾组织以及小肠组织中天然的钠 二羧酸协同转运蛋白 1.利用该抗体 ,首次证实了hNaDC1基因编码产物分布于人肾组织近端肾小管刷状缘 ,与大鼠钠 二羧酸协同转运蛋白 1(SDCT1)分布一致 .

Expression of Human Na~+/dicarboxylate Co-transporter 1 as Fusion Protein in E.coli and the Production of Its Antibody

The human Na +/dicarboxylate co\|transporter 1 (hNaDC1) mediates the transport of Krebs cycle intermediates, such as succinate, citrate , across the plasma membrane of mammalian cells. The cDNA fragments coding the deduced epitope of hNaDC1 (W138 Q219) were amplified by RT PCR. The PCR products were purified and digested with Eco R Ⅰ and Sal Ⅰ, then cloned into the expression vector pGEX 5X 1. The identity of the recombinant plasmid was verified by sequencing. The positive plasmid pGEX hNaDCL 6 was transformed into E.coli BL21, then cultured and induced with IPTG. The fusion protein GST hNaDC1 of 36 kD was produced at a level of 32% of the total cellular protein and purified by Glutathione Sepharose 4B affinity chromatography, then used as an immunogen to inoculate rabbits. The polyclonal antibody against the GST hNaDC1 fusion protein was raised and could specifically recognize the natural hNaDC1 located in the human kidney. Furthermore, the positive signal was also detected in the rat kidney and intestine. Using the antibody prepared in this experiments, it was confirmed that hNaDC1 localized in the brush border membrane of renal proximal tubule, which was similar to the localization of SDCT1.