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(英文)  

Constructing bicistronic eukaryotic expression plasmid containing human bone morphogenetic protein-2 labeled with green fluorescent protein

作　　者：苗军[1] 刘春蓉[2] 黄鸿超[1] 夏群[1] 石可松[1] 崔国盛 

机构地区：[1]天津市天津医院脊柱外科,天津市300211 [2]天津武警医学院病理学教研室,天津市200162 [3]沧州市第二人民医院,河北省沧州市061000

出　　处：《中国组织工程研究与临床康复》2008年第20期3984-3987,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30500516)~~

摘　　要：背景:腺病毒虽可携带骨诱导形成蛋白2基因转染靶细胞促进其分泌诱导形成蛋白2,但容易出现一些免疫应答反应,以质粒为载体的转染实验应具有良好的前景。目的:验证构建绿色荧光蛋白标记的人骨诱导形成蛋白2真核表达载体的可行性。设计:单一样本观察。单位:天津医院。材料: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(国家级)完成。含完整hBMP2基因片段的pcDNA3.1/CT-人骨诱导形成蛋白2质粒由李曦铭博士惠赠。双顺反子真核表达载体pSELECT-GFPzeo-MCS为Invivogen公司产品,Zeo购自Invivogen公司。pTA2-TEasy为鼎国生物技术有限公司产品,限制性内切酶BamHI和NheI、T4DNA连接酶(晶美生物工程有限公司),PCR上下游引物合成及测序(北京奥科生物技术有限责任公司)。方法:以重组质粒pcDNA3.1/CT-人骨诱导形成蛋白2为模板,结合已设计好的特异性引物,采用PCR方法亚克隆出人骨诱导形成蛋白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-人骨诱导形成蛋白2。主要观察指标:采用PCR法鉴定人骨诱导形成蛋白2序列,同时进行酶切及测序鉴定人骨诱导形成蛋白2是否克隆入pTA2-人骨诱导形成蛋白2重组质粒及真核表达载体pSELECT-GFPzeo-MCS中。结果:PCR获得长度约1216bp的目的片段,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BMP2cDNA序列(NM-001200)100%匹配。结论:成功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。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-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 a 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 1216 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 号：R329.47[医药卫生—人体解剖和组织胚胎学]