人血栓调节蛋白基因的表达及转基因小鼠的建立  

Expression of Human Thrombomodulin Gene and the Establishment of Transgenic Mice

作　　者：陈红英[1] 苟克勉[1] 王晓琳[1] 陈学进[1] 黄秀英[1] 孙方臻[1] 

机构地区：[1]中国科学院发育生物学研究所中科院分子发育生物学重点实验室,北京100080

出　　处：《中国兽医学报》2003年第2期115-118,共4页Chinese Journal of Veterinary Science

基　　金：国家"8 63"资助项目;国家自然科学基金重大项目(3 9993 43 0 )

摘　　要：用 PCR方法从人的基因组 DNA中扩增了人血栓调节蛋白 (h TM)基因 ,将其克隆到带有人 EF- 1α启动子的p EF- neo哺乳动物表达载体上 ,得到表达质粒 p EF- TM。在转染 p EF- TM的 COS- 1细胞中 ,h TM得到了瞬时表达 ,并且正确地定位到细胞膜上。用 p EF- TM转染猪内皮细胞 (PEC) ,经 G4 18筛选得到稳定转染的克隆。凝血活性测定结果显示 ,表达 h TM的 PEC凝血时间明显延长 ,表明其抗凝血能力增强。通过显微注射方法 ,得到了 1只 F0代 h TM转基因小鼠。 Southern杂交结果表明 ,该转基因小鼠整合了 9个拷贝的 p EF- TM DNA,并能将外源 DNA遗传给后代。It is supposed that overexpression of human anticoagulants such as human thrombomodulin(hTM) in a transplanted xenograft might significantly prolong the graft survival.In this study,hTM gene was amplified from human genomic DNA by PCR.It was inserted into pEF-neo mammalian expression vector containing human EF-1α promoter to create plasmid pEF-TM.In COS-1 cells transfected with pEF-TM,hTM was transiently expressed and anchored onto cell membrane.Stable transfection of porcine endothelial cells with pEF-TM resulted in expression of hTM,and transfected cells exhibited anticoagulation activity of prolongation of clotting time of human plasma.We obtained one transgenic mouse by microinjection with pEF-TM.Southern blot analysis showed that the founder animal had 9 copies of the transgene incorporated into its genomic DNA,and it could also transmit the foreign DNA to its offspring.

关 键 词：人血栓调节蛋白 转基因小鼠 异种移植 基因表达 器官移植 抗血栓 凝血酶 蛋白C 

分 类 号：Q78[生物学—分子生物学] R543[医药卫生—心血管疾病]