GFP示踪FLAG抗原表位标记BMP2转基因腺病毒穿梭质粒的构建  

Construction and identification of recombinant adenovirus shuttle plasimid expressing FLAG labeled BMP2 and traced by GFP

作　　者：张正[1] 李谌[1] 陈峻江[1] 刘丹平[1] 

机构地区：[1]辽宁医学院附属第一医院,辽宁锦州121001

出　　处：《山东医药》2011年第10期1-3,共3页Shandong Medical Journal

基　　金：辽宁省自然科学基金资助项目(20062199)

摘　　要：目的构建同时表达具有抗原表位FLAG标记的重组人骨形态发生蛋白2(rhBMP2)目的蛋白和示踪绿色荧光蛋白(GFP)报告分子的腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1。方法采用PCR技术对pcDNA3-BMP2携带的BMP2基因诱变,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。测序检测诱变情况,将诱变后的BMP2基因定向导入pShuttle CMV-IRES-hrGFP-1,将质粒分别转染人胚肾细胞HEK293A和兔骨髓间充质干细胞(MSCs)。通过限制性内切酶酶切图谱分析该质粒;采用荧光显微镜检查和免疫组化SP法测定HEK293A中的GFP和MSCs中的BMP2,行重组腺病毒穿梭质粒鉴定。结果质粒pShuttle CMV-BMP2+-IRES-hrGFP-1经双酶切鉴定图谱分析构建正确,HEK293A、MSCs中均有GFP和BMP2表达。结论 pShuttle CMV-BMP2+-IRES-hrGFP-1构建成功。Objective To construct a novel recombinant adenovirus shuttle plasimid expressing the BMP2 fused to FLAG epitope and green fluorescent protein（GFP） as a tracer protein of the recombinant adenovirus on the same transcript.Methods 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 PCR.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.The analysis of restricion map was adopted to identify the correct recombinants to monitor the expression of BMP2 and GFP in the MSCs and the HEK293A,the recombinants were transfected,fluorescence microscope and immunol histochemistry were employed.Rsults The plasimid（pShuttle CMV-BMP2＋-IRES-hrGFP-1） was contrcted correctly by two kinds of rectriction endnoucleases and sequence of the recombinant.The GFP and BMP2 were expressed in HEK293A and MSCs.Conclusion The adenovirus shuttle plasmid pShuttle CMV-BMP2＋-IRES-hrGFP-1 is constructed successfully.

关 键 词：骨形态发生蛋白2 重组腺病毒 质粒 绿色荧光蛋白 抗原表位 

分 类 号：Q812[生物学—生物工程]