含增强型绿色荧光蛋白rhBMP-2基因真核表达载体的构建  

Construction of a eukaryotic expression vector containing enhanced green fluorescence protein and recombinant human bone morphogenetic protein-2

作　　者：刘玉[1] 孙钦峰[2] 杨丕山[2] 杜芳[2] 

机构地区：[1]南京大学医学院附属口腔医院,江苏南京210008 [2]山东大学口腔医学院牙周病科,山东济南250012

出　　处：《上海口腔医学》2007年第6期627-631,共5页Shanghai Journal of Stomatology

基　　金：山东省自然科学基金(Y2006C125)

摘　　要：目的:构建含增强型绿色荧光蛋白的重组人骨形成蛋白-2(rhBMP-2)基因真核表达质粒。方法:采用PCR方法扩增rhBMP-2基因,并在两端添加合适的酶切位点;利用定向克隆技术将rhBMP-2基因和增强型绿色荧光蛋白(EGFP)基因分别插入到载体pIRES的上下游多克隆位点中,两者通过IRES连接,以防止融合表达;重组的pIRES-rhBMP2-EGFP在大肠杆菌DH5α内扩增后,通过酶切电泳鉴定和DNA测序证明质粒构建成功。结果:通过对重组质粒pIRES-rhBMP2-EGFP进行酶切鉴定以及DNA序列测定分析,证明真核表达重组质粒pIRES-rhBMP2-EGFP构建成功,开放阅读框架正确。结论:利用PCR、T/A克隆等分子生物学技术,可成功地将编码rhBMP-2的DNA片段和EGFP片段克隆到载体pIRES中,构建出重组子pIRES-rhBMP2-EGFP。PURPOSE： To construct recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP containing enhanced green fluorescence protein （EGFP） and recombinant human bone morphogenetic protein-2 （rhBMP-2）. METHODS： A pair of primers specific for amplifying the DNA fragment encoding rhBMP-2 were designed and synthesized. The targeted DNA fragment was amplified from pIRES-rhBMP2 by PCR.rhBMP-2 and EGFP were inserted to the proper sites of vector pIRES, and internal ribozyme entry site （IRES） sequence was between the genes coding for rhBMP-2 and EGFP. The recombinant plasmid pIRES-rhBMP2-EGFP was first propagated in E.coli DH5ot, and then was confirmed to contain hBMP2 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis. RESULTS： The construction of the recombinant eukaryotic dual-expression plasmid pIRES-rhBMP2-EGFP and the open reading frame were confirmed through restriction enzyme maping analysis and DNA sequencing. CONCLUSION： By PCR, T/A cloning, the cDNA fragment encoding rhBMP2 and EGFP fragment can be cloned into pIRES to construct the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP.

关 键 词：人骨形成蛋白-2(hBMP-2) 质粒 绿色荧光蛋白 

分 类 号：R78[医药卫生—口腔医学]