真核表达载体pcDNA3.0-Cx43的构建及其在骨髓基质干细胞中的表达

Construction and expressin of eukaryotic expression vector pcDNA3.0-Cx43 in mesenchymal stem cells

目的 :构建携带间隙连接蛋白connexin 4 3(Cx 4 3)cDNA的真核表达载体 ,转染骨髓基质干细胞 ,观察间隙连接蛋白Cx 4 3在骨髓基质干细胞中的过量表达 .方法 :目的基因Cx 4 3cDNA在大肠杆菌中扩增后连入真核表达载体pcD NA3.0 ,转染大鼠骨髓基质干细胞 ,用G4 1 8筛选得到Cx 4 3基因高表达的细胞 ;对照组为pcDNA3.0直接转染骨髓基质干细胞 .结果 :酶切电泳结果显示按设计构建的载体pcDNA3.0 Cx4 3,经G4 1 8筛选得到不同抗性的骨髓基质干细胞 ,免疫细胞化学显示有Cx 4 3蛋白的表达 .结论 :Cx 4 3可以在骨髓基质干细胞中表达 .

AIM: To construct a eukaryotic expression vector encoding full length connexin43 (Cx43) cDNA and to investigate its overexpression in mesenchymal stem cells (MSCs). METHODS: Full length Cx43 cDNA was amplified in E. coli JM109 and cloned into the eukaryotic expression vector pcDNA3.0. Stably transfected cell colonies were obtained by gene transfer and G418 screening and control cells were transfected with pcDNA3.0 and screened by using the same method. RESULTS: Electrophoresis results indicated that the recombinant vector was constructed correctly. Immunocytochemical analysis revealed an overexpression of Cx43 in pcDNA3.0 Cx43 transfected MSCs. CONCLUSION: The connexin43 can be expressed in MSCs.