pDsRed2-N1-SDF-1α真核表达载体的构建及在小鼠骨髓间充质干细胞中的表达  

Construction of a mouse pDsRed2-N1-SDF-1α eukaryotic expression vector and its expression in mouse bone marrow mesenchymal stem cells

作　　者：李明[1] 张世忠[1] 邹志浩[1] 郭燕舞[1] 卢凤飞[1] 姜晓丹[1] 寇盛斌[1] 卢国辉[1] 李振勇[1] 

机构地区：[1]南方医科大学珠江医院神经外科/广东神经外科研究所/广东省脑功能修复与再生重点实验室,广东广州510282

出　　处：《南方医科大学学报》2010年第3期439-442,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30772232)

摘　　要：目的构建pDsRed2-N1-SDF-1α真核表达载体并转染骨髓间充质干细胞(MSCs),观察其在MSCs内的表达。方法设计并合成SDF-1α基因引物,用RT-PCR法从小鼠平滑肌细胞中扩增出带有XhoI与EcoRI酶切位点的SDF-1α基因片段,将SDF-1α基因片段克隆到真核表达载体pDsRed2-N1上,酶切和测序鉴定。培养小鼠MSCs并Vimentin蛋白免疫荧光鉴定。通过脂质体将pDsRed2-N1-SDF-1α转染入小鼠MSCs。免疫荧光检测转染后的MSCs表达SDF-1α蛋白的情况。结果扩增出的基因片断大小与基因文库中已知的SDF-1α序列大小完全相符,酶切也得到目的基因片断,测序结果显示与已知的SDF-1α序列相同,成功构建pDsRed2-N1-SDF-1α真核表达载体。培养的细胞经Vimentin蛋白免疫荧光检测为阳性,pDsRed2-N1-SDF-1α表达载体转染到MSCs,24h后细胞免疫荧光检测可见SDF-1α融合蛋白表达。结论pDsRed2-N1-SDF-1α真核表达载体能够在小鼠MSCs中表达SDF-1α蛋白。Objective To construct the eukaryotic expression vector pDsRed2-N1-SDF-1α and observe its expression in the mouse bone marrow mesenchymal stem cells. Method SDF-1α gene sequence with XhoI, EcoRI restriction enzyme cutting site was amplified from the total RNA of mouse smooth muscle cells by reverse transcription-polymerase chain reaction （RT-PCR） and inserted into the eukaryotic expression vector pDsRed2-N1 encoding red fluorescent protein gene, and the insertion was verified by endonuclease digestion and DNA sequencing. Mouse bone marrow mesenchymal stem cells identified with immunofluorescence assay for vimentin expression were transfected with the constructed plasmid pDsRed2-N1-SDF-1α, and the expression of sdf-1α was detected using immunofluorescence assay. Results The DNA fragment amplified by PCR from the total RNA was identical to SDF-1α from the gene library, and an identical DNA fragment was also amplified from the recombinants. Sequence analysis confirmed the successful insertion of SDF-1α into the pDsRed2-N1 vector and the eukaryotic expression vector pDsRed2-N1-SDF-1α was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-1α expression 24 h after transfection with the recombinant vector. Conclusion The pDsRed2-N1-SDF-1α eukaryotic expression vector constructed is capable of expression of SDF-1α fusion protein in the mouse bone marrow mesenchymal stem cells.

关 键 词：SDF-1α蛋白 骨髓间充质干细胞 真核表达载体 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]