中文摘要：

目的：骨形态发生蛋白2是目前研究最为广泛、诱导成骨活性最强的骨形态发生蛋白之一。实验拟构建绿色荧光蛋白标记的人骨形态发生蛋白2（human Bone Morphogenetic Protein-2，hBMP2）真核表达载体，为在真核细胞的高效表达和基因治疗打下基础。方法：实验于2006-03／2007-03在天津医科大学卫生部激素与发育重点实验室完成。①实验材料：含完整人骨形态发生蛋白2基因片段的pcDNA3、1／CT-hBMP2质粒由李曦铭博士惠赠；双顺反子真核表达载体pSELECT-GFPzeo-MCS（Invivogen公司），Zeo（Invivogen公司）：pTA2-TEasy（鼎国生物技术有限公司）。②实验过程及评估：以重组质粒pcDNA3、1／CT-hBMP2为模板，结合已设计好的特异性引物，采用聚合酶链反应方法亚克隆出人骨形态发生蛋白2目的片段，将该片段分别与克隆载体pTA2-T-easy和双顺反子真核表达载体pSELECT-GFPzeo-MCS连接，转化入感受态DH5α细胞中，通过筛选得到含有绿色荧光蛋白的重组表达载体pSELECT-GFPzeo-hBMP2，并进行测序鉴定。结果：①聚合酶链反应获得长度约1216bp的目的片段，与预期片段相符。②经与克隆载体pTA2-T-easy和真核表达载体pSELECT-GFPzeo-MCS连接，筛选及序列分析后，证实所插入目的片段与GenBank检索的骨形态发生蛋白2cDNA序列（NM-001200）100％匹配。结论：成功构建含双顺反子绿色荧光蛋白人骨形态发生蛋白2真核表达载体。

英文摘要：

AIM： Bone morphogenetic protein-2 （BMP2） is presently one of widely used and actively BMP. This study aimed to construct green fluorescent protein （GFP）-labeled pSELECT-GFP zeo-hBMP2 eukaryotic expression vector. This result can provide basis for further research of hBMP2 highly effective expression and gene therapy. METHODS： Experiments were performed at the Key Laboratory of Hormone and Development of Ministry of Public Health of Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing complete human bone morphogenetic protein 2 （hBMP2） gene segment were presented by Doctor Li. Bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS （Invivogen, USA）, Zeo （Invivogen, USA）, and pTA2-T Easy （Dingguo Biotechnology Co., Ltd.） were used in this study. The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase chain reaction （PCR）. hBMP2 gene was inserted into pTA2-T-easy clone vector and pSELECT-GFPzeo-MCS eukaryotic expression vector, and then transferred into competence DH5α cells. After screening, pSELECT-GFPzeo-hBMP2 containing GFP was obtained and identified by sequence analysis. RESULTS： A 1 216 bp fragment was obtained by PCR, the same as expectant fragment. The hBMP2 fragment in pTA2-T-easy cloning vector and recombined pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis. The results were identical with that of reported hBMP2 sequence （Genebank NM-001200）. CONCLUSION： The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.