中国组织工程研究与临床康复2008，Vol.12Issue(20)：3984-3987,4.
构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体
Constructing bicistronic eukaryotic expression plasmid containing human bone morphogenetic protein-2 labeled with green fluorescent protein
摘要
Abstract
BACKGROUND: Bone morphogenetic protein-2(BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).DESIGN: Single sample observation.SETTING: Tianjin Hospital.MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2(R)-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5 α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPZeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.关键词
人骨形态发生蛋白2/真核表达载体/绿色荧光蛋白/组织构建分类
医药卫生引用本文复制引用
苗军,刘春蓉,黄鸿超,夏群,石可松,崔国盛..构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体[J].中国组织工程研究与临床康复,2008,12(20):3984-3987,4.基金项目
国家自然科学基金资助(30500516)the National Natural Science Foundation of China, No. 30500516 （30500516）
