转化生长因子β1基因真核表达质粒的构建及其在小鼠骨髓间充质干细胞中的表达

Construction of Eukaryotic Expression Vector for TGF-β1 and its Expression in Mouse Bone Marrow Mesenchymal Stem Cells

目的:构建转化生长因子β1(TGF-β1)表达载体,在骨髓间充质干细胞(BMSC)中表达。方法:以小鼠肺组织cDNA为模板,PCR扩增TGF-β1基因,并将其插入pCDH1-MCS1-EF1-copGFP载体质粒,转化感受态大肠杆菌DH5α,抽提质粒,经PCR和测序鉴定后转染BMSC,利用激光共聚焦显微镜和Western印迹对其表达进行检测。结果:经PCR及测序鉴定,构建入载体质粒的基因为TGF-β1基因,pCDH1-TGFβ1-EF1-copGFP重组质粒能在BMSC中表达。结论:构建了pCDH1-TGFβ1-EF1-copGFP重组质粒,且能表达于BMSC,为进一步研究TGF-β1影响间充质干细胞的生理功能奠定了基础。

Objective： To construct eukaryotic expression vector of the transforming growth factor β1 （TGF-β1）, and express the fusion protein TGF-β1-copGFP in mouse marrow mesenehymal stem cells（BMSC）. Methods： The full-length coding sequence of TGF-β1 was amplified from the eDNA of mouse lung sample by PCR, and was cloned into the Xba I/BstB I sites of the pCDH1-EF1-copGFP expression vector. After sequencing, the recombinant plasmid was transfected into BMSC and was observed by laser eonfocal microscopy and Western blot. Results： The results of PCR and sequencing showed that the recombinant plasmid pCDH1-TGFβ1-EF1-copGFP was constructed. What＇s more, the TGF-β1-eopGFP protein can expressed in BMSC. Conclusion： The recombinant plasmid pCDH1-TGFβ1-EF1-copGFP was constructed and it can express the protein TGF-β1 in BMSC, which provided a better understanding of the biological behavior of BMSC regulated by TGF-β1.