腺病毒穿梭质粒pShuttleCMV-BMP2^+-IRES-hrGFP-1的构建和鉴定  

Construction and Identification of Recombinant Adenovirus Shuttle Plasimid pShuttle CMV-BMP2^+-IRES-GFP-1

作　　者：张正[1] 刘丹平[1] 蒲勤[2] 郭韬[1] 张男[1] 

机构地区：[1]锦州医学院附属第一医院骨科,辽宁锦州121000 [2]第四军医大学生化教研室,陕西西安710032

出　　处：《锦州医学院学报》2005年第3期1-4,10,共5页Journal of Jinzhou Medical College

基　　金：国家自然基金资助项目 (编号 :3 0 0 70 75 7)

摘　　要：目的　构建腺病毒穿梭质粒pShuttleCMV -BMP2 + -IRES -hrGFP - 1,为构建表达具有抗原表位标记的骨形态发生蛋白2 (bonemorphogeneticprotein 2 ,BMP - 2 ) ,并同时表达绿色荧光蛋白(greenfluorescentpro tein ,GFP)报告分子的腺病毒真核细胞表达载体打下基础。方法　对目的基因供体质粒pcDNA3-BMP2携带的BMP2基因测序和序列内部存在的限制性内切酶识别位点进行分析,利用PCR (polymeraschainreaction ,PCR)技术对pcDNA3-BMP2携带的BMP2基因突变,以去除翻译终止密码子后的基因序列并添加新的酶切识别位点XhoⅠ。测序检测突变情况,将突变后的BMP2基因(BMP2 + 基因)定向连入腺病毒穿梭质粒pShuttleCMV -IRES -hrGFP - 1,通过限制性内切酶酶切图谱分析鉴定获得的重组质粒。结果　重组质粒经双酶切鉴定图谱正确。结论　成功构建了pShuttleCMV BMP2 + IRES hrGFP -1。Objective To clone bone morphogenetic protein 2(BMP2) gene into the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP-1 preparing for construction of a novel recombinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein(GFP) as a reporter on the same transcript. Methods The BMP2 gene contained in the plasimid of pcDNA3-BMP2 was sequenced, and the profile of restriction endonuclease sites existing in the sequence was analysed, then the base pairs behind the translation stop codon TAG were removed and a XhoⅠrestriction site was added following the 3’end of the mutant through polymeras chain reaction (PCR) mutagenesis technology. After being tested through sequencing, the mutant of BMP2 gene (BMP2^+ gene)was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP-1 by the directional cloning method. To identify the correct recombinants, the analysis of restriction map was adopted. Results The restriction map of the recombinant cleaved by two kind of restriction endonucleases accorded with theoretic analyses of pShuttle CMV-BMP2^+-IRES-hrGFP-1. Conclusions The adenovirus shuttle plasmid pShuttle CMV-BMP2^+-IRES-hrGFP-1 has been constructed successfully.

关 键 词：腺病毒穿梭载体 骨形态发生蛋白2(BMP-2) 

分 类 号：R373[医药卫生—病原生物学]