人MAdCAM-1真核表达载体的构建及其表达细胞株的建立

Construction and expression of human MAdCAM-1 eukaryotic expression vector

目的:建立表达人MAdCAM-1的细胞模型。方法:从pUC21/hMAdCAM-1质粒酶切下hMAdCAM-1全长cDNA片段,将其克隆于巨细胞病毒载体pCIneo中,构建出由CMV启动子控制的重组真核表达载体pCIneo-hMAd,经脂质体介导转染至CHO细胞,以G418筛选抗性克隆。结果:筛选出的阳性克隆细胞以免疫荧光和免疫酶标染色法检测,表明有人MAdCAM-1分子表达;其细胞裂解物经免疫印迹分析,证实约63 kD的新增蛋白带与抗 hMAdCAM-1抗体有特异性结合反应;淋巴细胞粘附实验结果提示表达产物具有一定的功能活性。结论:成功地获得了表达人MAdCAM-1分子的细胞株,为进一步研究其生物学功能及其作用机制奠定了基础。

To establish a cell line expressing human MAdCAM-1 molecular.Methods: hMAdCAM-1 cONA was achieved from plasmid pUC21/hMAdCAM-1 by restriction enzyme digestion. Hie recombinant eukaryotic expression vector pCIneo-hMAd was constructed and then transfected into CHO cells with lipofectamine. Results: G418-resistant clones and their expression products were identified by immunofluo-rescence、immunoenzymatic histochemistry、 western blot and lymphocytes adhesion assay. hMAdCAM-1 with relative molecule mass of 63 000 could be demonstrated in cell lysates of the pCIneo-hMAd transfected cells. Conclusion: A stable transfectant expressing hMAdCAM-1 is successfully established, which might provide the basis for further studying the biologic functions and their mechanism.