Mac-1-FP融合表达载体的构建、Mac-1-FP的表达与活性鉴定

分别构建表达BFP与CD1 1b的C末端、YFP与CD1 8的N末端相连接的融合蛋白的表达载体 ,并将二者转染至既无内源性Mac 1的表达同时又具有某些炎症反应信号转导系统的CHO细胞株进行表达Mac 1 FP .通过荧光显微镜观察到共转染后的CHO细胞可发出蓝色荧光和黄色荧光 ,应用Western印迹方法确定CD1 1b BFP与YFP CD1 8能够形成二聚体 ,采用流式细胞术检测确定PMA刺激Mac 1 FP可由胞浆内转位至膜上 ,测定PMA刺激前后的转染CHO细胞与其配基ICAM 1粘附活性的变化 ,证明转染CHO中的Mac 1 FP表达成功并具有野生型Mac 1的形成二聚体、膜转位、和配基ICAM 1相结合等功能 ,为进一步研究白细胞表面粘附分子Mac 1的α亚基CD1 1b、β亚基CD1 8在细胞内的走向及归宿创造了条件

Construction of Mac-1-FP Expression Vector, Expression of Mac-1-FP and Its Characterization

The mammalian cell expression vectors for Mac 1α subunit (CD11b) fused with blue fluorescent protein (BFP) at the carboxyl terminal or for Mac 1β subunit (CD18) fused with yellow fluorescent protein (YFP) at amino terminal were constructed to create recombinant plasmid of pCD11b BFP or pYFP CD18, respectively. Then both pCD11b BFP and pYFP CD18 were co transfected into CHO cells, a fibroblast like cell line, as a target cell within which there was some signal pathway related to inflammatory stimulation but without endogenous Mac 1. The blue and yellow fluorescence in co transfected CHO cells were observed under a fluorescence microscope, the picture of Western blot using antibody against CD11b and CD18 shown that the Mac 1 dimer consisting of CD11b BFP and YFP CD18 existing in the co transfected CHO cell and cytoplasm Mac 1 FP was capable of translocation to cell membrane while PMA stimulated co transfected CHO cells under flow cytometry. The adhesive rate of co transfected CHO cells with ligand ICAM 1 was increased after PMA stimulation. All the above results indicated that Mac 1 FP could be expressed in the co transfected CHO cells and exhibited some functions of wild type Mac 1, such as consisting dimmer, translocation from cytoplasm to membrane, adhesive activity with ligand ICAM 1. It may be useful for the study of the intercellular trafficking and fate of Mac 1.