人骨形成蛋白2真核表达载体的构建和体外表达  

Construction and expression in vitro of bone morphogenetic protein 2 eukaryotic experssion vector

作　　者：孙立[1] 田晓滨[1] 杨述华[2] 胡如印[1] 汪雷[1] 陆延盛[1] 张宇坤[2] 傅德皓[2] 

机构地区：[1]贵州省人民医院骨科,贵州省贵阳市550002 [2]华中科技大学同济医学院附属协和医院骨科,湖北省武汉市430022

出　　处：《中国组织工程研究与临床康复》2008年第50期9819-9822,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：贵州省医药攻关项目([2006]3053);贵州省科学技术基金项目([2007]2229)~~

摘　　要：背景:骨形态发生蛋白2(bone morphogenetic protein2,BMP-2)是已知的所有生长因子中对骨的形成作用最强的生长因子,被认为是最具有前途的骨诱导物质。目的:构建人骨形成蛋白2真核表达载体并观察其体外表达情况。设计、时间及地点:自身对照实验,于2005-07/2006-05在华中科技大学同济医学院分子生物中心实验室完成。材料:pcDNA3.1(+)载体由华中科技大学同济医学院左石博士惠赠;成骨肉瘤组织由华中科技大学同济医学院附属协和医院骨科提供。方法:从人成骨肉瘤细胞中提取细胞总RNA,利用反转录-聚合酶链反应方法扩增获得人BMP-2基因cDNA,将基因片断重组到pGEM-T质粒中构建pGEM-T-hBMP-2重组质粒载体,转化到大肠杆菌DH5α后筛选阳性克隆,利用限制性酶切和DNA序列分析鉴定重组质粒。分别用RcoRI和NotI双酶切pGEM-T-hBMP-2质粒和pcDNA3.1真核表达载体,将克隆载体中人骨形成蛋白2基因重组到pcDNA3.1真核表达载体,提取质粒作酶切电泳、聚合酶链反应鉴定及DNA测序后,用脂质体体外转染小鼠骨髓基质细胞,反转录-聚合酶链反应检测BMP-2的表达。主要观察指标:①人骨肉瘤细胞总RNA反转录-聚合酶链反应结果。②重组质粒pGEM-T-hBMP-2和pcDNA3.1-hBMP-2的构建和酶切鉴定。③BMP-2在小鼠骨髓基质细胞内的表达。结果:人骨肉瘤细胞总RNA经反转录-聚合酶链反应扩增后,获得1.2kb条带。经酶切电泳、聚合酶链反应鉴定及DNA测序证实实验成功克隆BMP-2基因,重组质粒pcDNA3.1-hBMP-2构建正确;该重组质粒能在体外培养的小鼠骨髓基质细胞中有效表达BMP-2。结论:实验成功克隆人骨形成蛋白2基因并构建了此基因的真核表达载体。BACKGROUND： Bone morphogenetic protein 2 （BMP2）, as a growth factor, has the strongest effect on bone formation in all growth factors that have been known. It is considered as a bone-inducing substance with most desirable prospects. OBJECTIVE： To construct a human BMP2 gene eukaryotic expression vector, and observe its expression in vitro. DESIGN, TIME AND SETTING： Self-control experiment was performed at Central Laboratory of Molecular Biology, Tongji Medical College of Huangzhong University of Science and Technology from July 2005 to May 2006. MATERIALS： PcDNA3.1（＋） plasmid was a gift from Doctor Zuo Shi of Tongji Medical College of Huazhong University of Science and Technology. Human osteosarcoma tissues were provided by Department of Orthopedics, Wuhan Union Hospital, Tongji Medical College of Huangzhong University of Science and Technology, METHODS： The total RNA was extracted from human osteosarcoma cells. The human BMP2 cDNA was amplified by RT-PCR and inserted into pGEM-T vector to construct BMP2 vector. After BMP2 vector had been transformed into E. coli DH-5α, the po.sitive clones were screened out. Then the recombinant plasmids were identified by restriction enzyme digestion and DNA sequencing, By digestion of pGEM-T- hBMP-2 plasmid with RcoR I and pcDNA3.1 eukaryotic expression vector with Not I, the BMP2 cDNA in the pGEM-T cloning vector was inserted into the pcDNA3.1 eukaryotic expression vector. The recombinant plasmids were identified by restriction fragments electrophoresis, PCR and DNA sequencing. Mouse bone marrow stromal cells （BMSCs） were transfected with the vectors using lipofecarmin reagents. The expression of BMP-2 was detected by RT-PCR and immunohistochemical analysis. MAIN OUTCOME MEASURE： （1）Results of the total RNA extracted from human osteosarcoma cells amplified by RT-PCR; （2） The construction and identification by restriction fragments electrophoresis of pGEM-T-hBMP-2 and pcDNA3.1-hBMP-2; （2） Expression of BMP-2 in mouse BMSCs. RE

关 键 词：骨形成蛋白2 真核表达载体 基因克隆 

分 类 号：R318[医药卫生—生物医学工程]