CAJ | 학술논문

目的构建pDsRed2-Nl-SDF-lα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Xhol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-Nl-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,24 h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.
목적 구건pDsRed2-Nl-SDF-lα진핵표체재체병전염골수간충질간세포(MSCs),관찰기재MSCs내적표체.방법 설계병합성SDF-1α기인인물,용RT-PCR법종소서평활기세포중확증출대유Xhol여EcoRI매절위점적SDF-1α기인편단,장SDF-1α기인편단극륭도진핵표체재체pDsRed2-N1상,매절화측서감정.배양소서MSCs병Vimentin단백면역형광감정.통과지질체장pDsRed2-N1-SDF-1α전염입소서MSCs.면역형광검측전염후적MSCs표체SDF-1α단백적정황.결과 확증출적기인편단대소여기인문고중이지적SDF-1α서렬대소완전상부,매절야득도목적 기인편단,측서결과 현시여이지적SDF-1α서렬상동,성공구건pDsRed2-Nl-SDF-1α진핵표체재체.배양적세포경Vimentin단백면역형광검측위양성,pDsRed2-N1-SDF-1α표체재체전염도MSCs,24 h후세포면역형광검측가견SDF-1α융합단백표체.결론 pDsRed2-N1-SDF-1α진핵표체재체능구재소서MSCs중표체SDF-1α단백.
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-lα gene sequence with Xhol, 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-lα 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-lα was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-lα 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.