纤溶酶原激活物抑制物-1/绿色荧光蛋白融合基因表达载体的构建及其在系膜细胞的表达

Construction of PAI 1 /GFP fusion gene expression vector pCMX GFP/PAI 1 and its expression in rat mesangial cells

目的 以绿色荧光蛋白（ Ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｃｅ ｐｒｏｔｅｉｎ ， Ｇ Ｆ Ｐ） 为报告基因，构建纤溶酶原激活物抑制物１（ Ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｔｏｒｉｎｈｉｂｉｔｏｒ１ ， Ｐ Ａ Ｉ１） 与 Ｇ Ｆ Ｐ融合基因的真核表达质粒，在活细胞状态下观察 Ｐ Ａ Ｉ１／ Ｇ Ｆ Ｐ融合蛋白在大鼠系膜细胞中表达 。方法 利用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术，从含有编码人类 Ｐ Ａ Ｉ１（ｈ Ｐ Ａ Ｉ１） 全长ｃ Ｄ Ｎ Ａ 序列的质粒ｐ Ｕ Ｃ１９ Ｐ Ａ Ｉ１ 中扩增出ｈ Ｐ Ａ Ｉ１ 的编码区全长ｃ Ｄ Ｎ Ａ 序列，定向克隆至真核表达载体ｐ Ｃ Ｍ Ｘ Ｇ Ｆ Ｐ。利用脂质体为转染载体，将表达质粒转染至体外培养的大鼠系膜细胞。利用 Ｎｏｒｔｈｅｒｎ 杂交、 Ｗｅｓｔｅｒｎ 印迹、荧光显微镜直接观察以及纤维蛋白平板等方法检测 Ｐ Ａ Ｉ１／ Ｇ Ｆ Ｐ融合基因在大鼠系膜细胞的表达。结果 转染组大鼠系膜细胞 Ｐ Ａ Ｉ１ ｍ Ｒ Ｎ Ａ 表达、细胞培养上清 Ｐ Ａ Ｉ１ 活性均较对照组明显增高。大鼠系膜细胞表达的融合蛋白 Ｐ Ａ Ｉ１／ Ｇ Ｆ Ｐ 对尿激酶型纤溶酶原激活物（ｕ Ｐ Ａ） 有明显的抑制活性，并能在荧光显微镜下激发绿色荧光。?

Objective To construct PAI 1/GFP fusion gene eukaryotic expression vector pCMX PAI 1 GFP and investigate PAI 1/GFP gene expression in rat mesangial cells.Methods Human PAI 1 cDNA was amplified from pUC19 PAI 1 by polymerase chain reaction method and inserted into mammalian expression plasmid pCMX GFP. Using lipofectin method, the recombinant expression plasmid pCMX PAI 1 GFP was transfected into rat mesangial cells.Results PAI 1/GFP gene was demonstrated by Northern blot analysis and Western blot analysis to be transcripted and translated in rat mesangial cells. PAI 1/GFP gene could express a specific protein. The recombinant PAI 1/GFP had significant inhibition activity to urokinase type plasminogen activator and displayed autonomous fluorescence. Conclusion PAI 1/GFP fusion protein,expressed by rat mesangial cells shows a significant PAI 1 activity with fluorescence.