论文部分内容阅读
目的探讨人骨肉瘤细胞U2-OS表达重组人骨形态发生蛋白-7(recombinant human bone morphogenetic protein-7,rhBMP-7)的可行性。方法将hBMP-7成熟肽基因片段克隆至载体pcDNA3.1上,构建真核表达质粒pcDNA3.1-rhBMP-7。利用Lipofectamine 2000将重组质粒转染至U2-OS细胞中,采用实时荧光定量PCR及Western blot法检测细胞中hBMP-7基因mRNA和rhBMP-7的表达。结果真核组表达质粒pcDNA3.1-rhBMP-7经NdeⅠ和EcoRⅤ双酶切及测序鉴定证明构建正确。pcDNA3.1-rhBMP-7质粒转染的U2-OS细胞中hBMP-7基因mRNA和rhBMP-7的表达水平均明显高于pcDNA3.1载体转染的细胞(P<0.05)。结论成功构建了hBMP-7基因的重组表达质粒,并在U2-OS细胞中成功表达,为进一步研究hBMP-7的制备方法和建立新的表达系统奠定了基础。
Objective To investigate the feasibility of recombinant human bone morphogenetic protein-7 (rhBMP-7) expressed by human osteosarcoma cell line U2-OS. Methods The hBMP-7 mature peptide gene fragment was cloned into vector pcDNA3.1 to construct eukaryotic expression plasmid pcDNA3.1-rhBMP-7. The recombinant plasmids were transfected into U2-OS cells by Lipofectamine 2000. The expression of hBMP-7 mRNA and rhBMP-7 mRNA were detected by real-time fluorescence quantitative PCR and Western blot. Results Eukaryotic expression plasmid pcDNA3.1-rhBMP-7 by Nde Ⅰ and EcoR V double digestion and sequencing proved that the construction is correct. The expression levels of hBMP-7 mRNA and rhBMP-7 in U2-OS cells transfected with pcDNA3.1-rhBMP-7 were significantly higher than those transfected with pcDNA3.1 (P <0.05). Conclusion The recombinant plasmid expressing hBMP-7 gene was successfully constructed and successfully expressed in U2-OS cells, which laid the foundation for further study on the preparation of hBMP-7 and establishment of a new expression system.