人骨形态发生蛋白-2真核表达质粒的制备、转染及表达

Research of construction,transfection and expression of human bone morphogenetic proteins factor-2 eukaryotic plasmid

目的制备含人骨形态发生蛋白-2(hBMP-2)基因的真核表达质粒,获得可稳定高效表达BMP-2的兔骨髓基质细胞,为进一步研究BMP-2在骨牵张区的成骨作用作准备。方法制备携带有hBMP-2的全长真核表达质粒pcDNA3.1-BMP2。通过脂质体法将所得到的重组真核质粒转染到兔骨髓基质细胞中,用G418进行梯度筛选,从而得到能够稳定表达hBMP-2的细胞克隆。采用逆转录聚合酶链反应(RT-PCR)检测骨髓基质细胞(MSCs)hBMP-2 mRNA的表达。结果重组质粒通过EcoRI酶切后,电泳图示约1.5 kb的hBMP-2的目的片段及5.5 kb的载体片段,经测序显示核苷酸序列正确无误。经RT-PCR检测hBMP-2在转录水平mRNA的表达情况,提示转染组hBMP-2的mRNA量较空白对照组明显增加。结论成功制备了含hBMP-2基因的真核表达质粒,转染后获得了高效表达hBMP-2的兔骨髓基质细胞。

Objective Constructing the eukaryotic expression plasmid of human bone morphogenetic proteins factor-2 （hBMP-2）,and transfering it into rabbit marrow stromal cells （MSCs） to make a stable expression, so as to investigate the function of hBMP-2 in distraction osteogenesis, especially in the bone tissue engineering. Methods The hBMP-2 cDNA was amplified and cloned into the eukaryotic plasmid pcDNA3.1 and screened with Escberichia coli JM109. The recombinant plasmid was transfered into the rabbit marrow stmmal cells by lipofectamine, and the positive cell clones were selected with G418. Expression of hBMP-2 mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR）. Results The new recombinant plasmid was digested with EcoRI, and the electrophoresis of the digested products showed 1.5 kb and 5.5 kb fragments. It was showed by the RT-PCR method that the hBMP-2 gene transfected MSCs prominently elevated mRNA expression of hBMP-2. It was confirmed that hBMP-2 gene could be expressed effectively in MSCs transfected with the recombinant plasmid. Conclusion The pcDNA3.1- hBMP-2, a eukaryotic expression plasmid of hBMP-2 gene, has been constructed. The hBMP-2 gene could be expressed effectively in rabbit marrow stromal cells transfected with the recombinant plasmid.