YFP-CD18融合蛋白在U937细胞株中的表达与鉴定  

Characterization of CD18- YFP Fusion Protein Expressed in U937 Cellline

作　　者：严鸣[1] 毛积芳[2] 钟纪根[1] 何健[3] 朱秋峰[4] 徐仁宝[1] 

机构地区：[1]基础医学部病理生理学教研室 [2]基础医学部生物化学与分子生物学教研室 [3]长海医院急救科 [4]长征医院麻醉科,第二军医大学,上海200433

出　　处：《生物技术》2003年第4期4-6,共3页Biotechnology

基　　金：国家自然科学基金项目 ("Mac - 1在白细胞内的走向及归宿的动态定量研究" ;30 0 0 0 0 68);国家自然科学基金重大项目 ("单分子Mac - 1的构象与亲和力的实时定量动态研究" ;30 2 91 70 5)

摘　　要：目的 :构建并表达Mac - 1的β链CD18与黄色荧光蛋白 (YFP)的融合蛋白YFP -CD18,为进一步直视研究Mac- 1在白细胞内的分布、走向及归宿提供物质条件。方法 :首先将CD18的全长cDNA与pEYFP -C1进行酶切 ,构建YFP与CD18的N末端连接的表达载体 ,并将其转染至U937细胞株中进行表达。通过荧光显微镜观察转染成功后的U937细胞黄色荧光是否存在 ,以及流式细胞术检测确定PMA刺激后YFP -CD18活化后可否由胞浆内转位至膜上和测定PMA活化前后转染的U937细胞与其配基ICAM - 1粘附活性的变化等方面来进行鉴定。结果 :经酶切鉴定 ,pYFP -CD18构建正确 ,转染U937细胞株后 ,可见YFP -CD18融合蛋白发出的黄色荧光。结论 :构建完成YFP -CD18融合基因并可以在U937细胞株中进行表达。Objective:To construct the expressive vector of pYFP-CD18 and to express fusion protein YFP-CD18 in U937 cellline in order to provide foundational preparation for advanced researching in intracellular distribution and dynamic trafficking of Mac-1.Methods:Full length CD18 cDNA was inserted into the MCS of pEYFP-N1 vector to construct the expression vector of pYFP-CD18 after they were digested by restriction endonuclease.Then,the U937 cellline was transfected with recombinant vector containing fusion gene and expressed fusion proteins YFP-CD18,which were visible with a fluorescence microscope and were measured by flow cytometry and were assayed with ligand ICAM-1.Results:Restriction endonuclease digestive identification was right for recombinant expression vector pYFP-CD18.Yellow fluorescence from fusion protein could be seen in U937 cells transfected with plasmid pYFP-CD18.Conclusions:Recombinant expressive vector of pYFP-CD18 was constructed and expressed in U937 cellline successfully.

关 键 词：YFP CD18 融合蛋白 U937细胞株 表达 黄色荧光蛋白 

分 类 号：Q786[生物学—分子生物学] Q51