人钠依赖性二羧酸转运蛋白2(hSDCT2)的组织表达谱分析

利用DNA重组技术 ,构建重组表达质粒pGEX hSDCT2 .IPTG诱导其表达后 ,采用谷胱甘肽 Sepharose 4B亲和层析 ,获得纯化的谷胱甘肽S 转移酶 (GST) hSDCT2重组融合蛋白 .以此为免疫原免疫兔制备GST hSDCT2融合蛋白抗体 .多组织Northern印迹法结果显示 ,3 6kb的hSDCT2基因转录产物 ,在心、骨骼肌、胸腺、小肠、肺和外周血白细胞等组织中几乎不表达 ,在脑、结肠、脾、肝和胎盘中仅有少量表达 ,但在肾脏中大量表达 ;并且在肾脏和脾脏中还存在着另一种约 4 3kb的转录产物 .Western印迹法证实 ,hSDCT2蛋白以类似方式于上述组织表达 .免疫组化双重染色结果发现 ,与分布于近端肾小管刷状缘的hSDCT1不同 ,hSDCT2主要分布于近端肾小管的基底膜侧 .这些结果为进一步研究人钠依赖性二羧酸转运蛋白 2的结构和功能奠定了基础 .     

T-cell restricted and unrestricted expression of transfected human interleukin-2 gene: phorbol ester- and calcium-inducible versus constitutive expression

Interleukin-2 (IL-2) gene expression is tightly controlled and generally limited to antigenic stimulation of T cells. To study the cell-specific expression of the IL-2 gene, we transfected the intact human IL-2 gene, including 2.0 kb of 5' and 0.3 kb of 3' flanking sequences, into mouse NIH-3T3 fibroblasts and BFS lymphoma T cells and into human epithelial HeLa cells. Stable transformants (NIH-3T3,HeLa and BFS cells) carried an intact transfected IL-2 gene and constitutively expressed cytoplasmic human IL-2 mRNA which was not detected in vector-transfected cells. Constitutive expression of IL-2 mRNA in human IL-2 gene-transfected NIH-3T3 and HeLa cells was associated to the secretion of bioactive IL-2 protein, while no IL-2 production was observed in untransfected or vector-transfected cells. Cytoplasmic IL-2 mRNA observed in transfectants was larger (1.4 kb) than endogenous IL-2 mRNA of human T cells, although smaller than RNA containing unspliced intact introns. No alternative promoters or polyadenylation signals were used by these cells, but some intronic sequences were present in the 1.4 kb mRNA. Phorbol ester and calcium ionophore did not modulate the expression of the transfected IL-2 gene in NIH-3T3 and HeLa cells, while these agents increased its expression in transfected BFS lymphoma T cells. We conclude that when transfected into lymphoid and non-lymphoid cells the intact human IL-2 gene is constitutively expressed, while its phorbol ester/calcium-mediated inducible expression is restricted to T cells. This suggests that the constitutive and inducible expression of the IL-2 gene can be dissociated and are presumably subjected to separate regulatory pathways.