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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 pDsRed2-N1-SDF-1α eukaryotic expression vector and transfect it into bone marrow mesenchymal stem cells (MSCs) to observe its expression in MSCs. Methods SDF-1α gene primers were designed and synthesized. The SDF-1α gene fragment with XhoI and EcoRI restriction sites was amplified from mouse smooth muscle cells by RT-PCR. The SDF-1α gene fragment was cloned into eukaryotic expression vector The vector pDsRed2-N1 was digested and sequenced. MSCs were cultured and identified by immunofluorescence with Vimentin protein. PDsRed2-N1-SDF-1α was transfected into mouse MSCs by liposomes. The expression of SDF-1α protein in transfected MSCs was detected by immunofluorescence. Results The size of the amplified gene fragment was completely consistent with the known size of SDF-1α in the gene library, and the target gene fragment was also obtained by digestion. The sequencing result showed that the same sequence as the known SDF-1α gene was successfully constructed and the pDsRed2- SDF-1α eukaryotic expression vector. The cultured cells were detected by immunofluorescence with Vimentin protein. The pDsRed2-N1-SDF-1α expression vector was transfected into MSCs, and the expression of SDF-1α fusion protein was detected by immunofluorescence. Conclusion The pDsRed2-N1-SDF-1α eukaryotic expression vector can express SDF-1α protein in mouse MSCs.