人骨形成蛋白7真核表达质粒的构建及其在骨髓基质细胞的表达
骨形态发生蛋白质类;,基因;,真核细胞;,骨髓细胞,,骨形态发生蛋白质类;,基因;,真核细胞;,骨髓细胞,1材料与方法,2结果,3讨论,参考文献：
摘要: 目的 利用基因工程原理构建pIRES2EGFPhBMP7真核表达质粒并检测其在Beagle犬骨髓基质细胞(BMSC)的表达。 方法 利用DNA重组技术,将hBMP7片段克隆到真核表达载体pIRES2EGFP中,EcoRⅠ单酶切、PCR及序列分析鉴定获得的重组质粒;脂质体介导的方法转染Beagle犬BMSC;免疫组织化学染色检测hBMP7基因的表达。结果 hBMP7被定向插入到真核表达载体pIRES2EGFP中,转染BMSC 24 h后可观察到转染的BMSC表达绿色荧光蛋白,48 h后转染效率达到31%。免疫组织化学染色证实重组pIRES2EGFPhBMP7在BMSC中的表达。结论 利用基因工程原理成功构建真核表达质粒pIRES2EGFPhBMP7,并可在BMSC中表达。关键词: 骨形态发生蛋白质类; 基因; 真核细胞; 骨髓细胞
Construction of Human Bone Morphogenetic Protein7 into Eukaryotic Expressive Plasmid and Its Expression in Bone Marrow Stromal Cells
Li Yanfen, Yan Fuhua, Jiang Yiping, Luo Kai, Zhao Xin
1.College of Stomatology, Fujian Medical University, Fuzhou 350002, China
2.Center of Cell and Developmental Biology, Fujian Medical University, Fuzhou 350004, China
ABSTRACT: Objective To construct pIRES2EGFPhBMP7 eukaryotic expression plasmid by genetic engineering and observe its expression in bone marrow stromal cells(BMSC) of Beagle dog. Methods The hBMP7 cDNA was inserted into the eukaryotic expression vector pIRES2EGFP.The recombinant plasmid was identified by restriction enzymolysis(EcoRⅠ), PCR analysis and DNA sequencing. Liposomemediated gene transfer method was used to transfect bone marrow stromal cells(BMSC).The immunohistochemical staining was employed to observe the expression of hBMP7 gene. Results The hBMP7 gene was cloned into the vector successfully. The transfected BMSC expressing green fluorescene protein were observed under fluorescence microscope. The gene transfection efficiency was 31% after 48 hours. The immunohistochemical staining showed the expression of hBMP7 in BMSC.Conclusion pIRES2EGFPhBMP7 eukaryotic expression plasmid, which contained the full length sequence of the hBMP7 gene could be transfect into BMSC. BMSC expressed the hBMP7 protein. ......
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