内皮特异性表达人血栓调节蛋白的载体构建及体外表达  

Construction and In vitro Expression of Vectors Which Specifically Express Human(Homo sapiens) Thrombomodulin in Pig(Sus scrofa)Endothelium

作　　者：宋小凤[1] 石宁宁[1] 冯冲[1] 魏静 周艳荣[2] 陈红星[2] 潘登科[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室,北京100193 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《农业生物技术学报》2015年第12期1597-1603,共7页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(No.2011CBA01004)

摘　　要：血栓调节蛋白(thrombomodulin,TBM)是血管内皮细胞表达的一种膜蛋白,在凝血过程中起到关键作用。已有研究表明,异种移植时由于分子不匹配造成猪(Sus scrofa)TBM不能有效地调节凝血而形成血栓,而在猪的血管内皮细胞表达人TBM可能有助于调节凝血。本研究利用猪源TBM启动子调控人(Homo sapiens)TBM基因(Human thrombomodulin,h TBM),采用基因抓捕的方法获得了7和9 kb猪血栓调节蛋白(Pig thrombomodulin,p TBM)启动子,构建两种表达h TBM的载体,命名为p7K(p TBM)-h TBM和p9K(p TBM)-h TBM。体外转染猪内皮细胞、耳成纤维细胞及肾细胞,通过RT-PCR、q RT-PCR和Western blot检测其特异性表达。RT-PCR显示,7和9 kb两种启动子都能特异调控h TBM在血管内皮表达;q RT-PCR和Western blot显示,9 kb启动子的启动活性高于7 kb(P<0.05)。本研究表明,9 kb p TBM启动子能够驱动h TBM在血管内皮特异性高效表达,为创建血管内皮特异高表达人抗凝血因子的转基因猪提供了基础资料,有助于克服异种移植分子不匹配出现的凝血失调。Clotting disorders by molecular mismatch is one of the remaining critical issues in pig-to-human xenotransplantation. Thrombomodulin（TBM） is an endothelial transmembrane glycoprotein that plays a critical role in the control of coagulation. Studies demonstrated that pig（Sus scrofa） TBM can＇t effectively regulate blood clotting and thrombosis if the molecules do not match. Thus, the expression of exogenous human TBM in pig vascular endothelial cells may be helpful for overcoming the molecular incompatibilities. In this study, the 7and 9 kb promoter of pig TBM gene were obtained by gap-repair method, and correspondingly 2 expression vectors of h TBM（p7K（p TBM）-h TBM, p9K（p TBM）-h TBM） were constructed. In order to validate and compare the specificity and efficiency of the 2 vectors, both of them were transfected into endothelial cells, ear fibroblast and kidney cells of Wuzhishan miniature pig. The transfected cells were cultured for 48 h and then harvested for RT-PCR and Western blot analysis. The results of RT-PCR indicated that both the 7 and 9 kb promoters could specifically regulate the expression of hT BM in vascular endothelium; qR T- PCR and Western blotting analysis showed that the activity of 9 kb promoter was higher than the that of 7 kb counterpart（P〈0.05）. Altogether, the results in vitro demonstrated that the 9 kb TBM promoter of pig was a more suitable candidate for endothelial cell specific expression, based on the fact that it could drive the expression of hT BM at higher efficiency. This work lays the foundation for the future transgenic pig efficiently express anti-coagulation factor in its vascular endothelium, which will solve the clotting disorders caused by a molecular mismatch in xenotransplantation.

关 键 词：血栓调节蛋白(TBM) 血管内皮 特异性表达 凝血失调 猪 

分 类 号：Q513.2[生物学—生物化学]