pcDNA3.1(+)-CTGF真核表达质粒构建及其在人成骨样细胞SaOS-2细胞中的表达  

Construction of eukaryotic expression plasmid of pcDNA3.1(+)-CTGF and its expression in human osteoblast-like cells SaOS-2

作　　者：马科锋 杨树广 刘少君 MA Ke-feng;YANG Shu-guang;LIU Shao-jun(Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《中国应用生理学杂志》2019年第6期573-576,I0004,共5页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(81370051)。

摘　　要：目的:构建结缔组织生长因子(CTGF)的pcDNA3.1(+)真核表达质粒(pcDNA3.1(+)-CTGF),并检测其在人成骨样细胞SaOS-2中的表达,为进一步研究CTGF基因在骨发育和骨修复中的机制提供技术支撑。方法:采用PCR方法体外克隆CTGF基因全序列,将其用同源重组技术连接到线性pcDNA3.1(+)载体上,构建pcDNA3.1(+)-CTGF真核表达质粒,并对该质粒进行测序鉴定;鉴定无误后转染至SaOS-2细胞中,观察其48 h的表达情况。结果:基因测序证实pcDNA3.1(+)-CTGF真核表达重组质粒构建成功,与对照组相比,转染SaOS-2细胞48 h后的CTGF表达水平显著上调,达到对照组的4.8×105倍(P<0.01)。结论:成功构建了pcDNA3.1(+)-CTGF真核表达质粒,并能在人成骨样细胞SaOS-2中稳定表达,为深入研究CTGF基因对骨生成的调控机制奠定了基础。Objective:To construct pcDNA3.1(+)eukaryotic expression plasmid of connective tissue growth factor(CTGF),and detected its expression in human osteoblast-like cells SaOS-2,which provides a technical support for further research on the mechanism of CTGF gene in bone development and bone repair process.Methods:The whole sequence of CTGF gene was cloned in vitro by polymerase chain reaction(PCR)method and connected to the linear pcDNA3.1(+)vector for constructing pcDNA3.1(+)-CTGF eukaryotic expression plasmid by homologous recombination technology.The plasmid was identified by sequencing.After identification,it was transfected into SaOS-2 cells and its expression was detected at 48 h.Results:pcDNA3.1(+)-CTGF eukaryotic expression recombinant plasmid was successfully constructed,which was confirmed by sequencing.Compared with the control group,CTGF expression level was significantly up-regulated after transfection of SaOS-2 cells for 48 h,up to five times as much as the control group.Conclusion:pcDNA3.1(+)-CTGF eukaryotic expression plasmid was successfully constructed and could be stably expressed in human osteoblasts-like cell SaOS-2,which laid a foundation for further study on the regulatory mechanism of CTGF gene on bone formation.

关 键 词：结缔组织生长因子(CTGF) 人成骨样细胞 质粒构建 真核表达 骨生成 

分 类 号：Q813.1[生物学—生物工程]