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*

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。

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

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.