真核表达载体pCMV-(Kozak) TFPI的构建及其在内皮细胞中的表达  

Construction and Expression in HUVEC of Eukaryotic Expressed Plasmid Vector pCMV-(Kozak) TFPI

作　　者：厉泉[1] 张凯伦[1] 龚永生[1] 徐鹏[1] 郭超[1] 祖育昆[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心外科,湖北武汉430022

出　　处：《中国微循环》2007年第4期240-243,共4页Journal of Chinese Microcirculation

基　　金：国家自然科学基金项目(30571838)

摘　　要：目的构建真核表达载体pCMV-(Kozak)TFPI并检测其在内皮细胞中的表达,为冠状动脉旁路移植和经皮冠状动脉内成形术中转染血管内皮细胞抗凝治疗作了实验和理论的探索。方法用RT-PCR的方法提取人组织因子途径抑制因子(TFPI)基因,并引入Kozak序列,将(Kozak)TFPI亚克隆入pCMV质粒中。在阳离子脂质体介导下,转染人脐静脉内皮细胞(HUVEC)。RT-PCR、免疫荧光和Western blot检测HUVEC中外源TFPI基因mRNA和蛋白的表达。结果(Kozak)TFPI成功克隆入pC-MV中。RT-PCR、免疫荧光和Western blot检测到外源TFPI基因mRNA和蛋白的表达。结论成功构建了pCMV-(Kozak)TFPI真核表达载体,并表达出相应的蛋白。Objective To construct eukaryotic expressed plasmid vector pCMV-（Kozak） TFPI and detect expression of it in endotheliocytes, further study anticoagalation effect in vessels of TFPI gene transfection in coronary artery bypass graft and percutaneous transluminal coronary angioplasty. Methods The full length human tissue factor pathway inhibitor（TFPI） gene was extracted by RT-PCR, and the Kozak sequence was led in TFPI gene. Subsequently, the（Kozak） TFPI sequence was inserted in eukaryotic expressed plasmid vector pCMV. Human umbilical vein endotheliocytes（HUVEC） were transfected with cationic liposome containing the plasmid vector pCMV-（Kozak） TFPI. RT-PCR, Immunofluorescence and Western blot were applied to detect expression of TFPI mRNA and protein in HUVEC. Results The （Kozak） TFPI sequence was cloned into eukaryotic expressed plasmid vector pCMV. RT-PCR, Immunofluorescence and Western blot detected expression of TFPI mRNA and protein in HUVEC. Conclusion The eukaryotic expressed plasmid vector pCMV-（Kozak） TFPI was constructed, and TFPI protein was expressed successfully in HUVEC.

关 键 词：冠状动脉旁路移植术 经皮冠状动脉内成形术 组织因子途径抑制因子 基因 载体 转染 

分 类 号：Q785[生物学—分子生物学]