真核表达载体pcDNA3.1(+)-vIL-10的构建及其在SD大鼠骨髓基质干细胞的表达

目的构建携带免疫调节因子vIL-10c DNA的真核表达载体,转染至SD大鼠骨髓基质干细胞,观察免疫调节因子vIL-10在骨髓基质干细胞的表达。方法PCR扩增原核载体PET-28α/vIL-10,获得目的基因vIL-10,目的基因在大肠杆菌中扩增后连入真核表达载体Pc DNA3.1(+),转染大鼠骨髓基质干细胞。用G418筛选得到vIL-10高表达的细胞,对照组为pcDNA3.1(+)直接转染的骨髓基质干细胞。结果经PCR、酶切、测序鉴定显示按设计构建的载体pc DNA3.1(+)-vIL-10,已成功转染骨髓基质干细胞。RT-PCR检测到510bp目的片段vIL-10在骨髓基质干细胞表达,SDS-PAGE电泳表明在骨髓基质干细胞有vIL-10蛋白的表达。结论vIL-10 mRNA和蛋白在骨髓基质干细胞中表达。

Construction and expression of eukaryotic expression vector pcDNA3.1 (+) /vIL-10 in mesenchymal stem cells of rat

Objective To construct a eukaryotic expression vector encoding vIL-10 cDNA and to investigate its expression in mesenchymal stem cells（MSCs） of rat.Method The encoding region of vIL-10 was cloned with PCR from prokaryotic vector PET-28α /vIL-10 and amplified in E.coli JM109 and cloned into the eukaryotic expression vector pcDNA3.1（＋）.The transfected cell colonies were obtained by gene transfer and G418 screening,and control cells were transfected with pcDNA3.1（＋） and screened using the same method.Results PCR,double enzyme-digestion and DNA sequencing showed that the recombinant vector pcDNA3.1（＋）/vIL-10 was constructed correctly and transfected into MSCs successfully.A band of about 510 bp mRNA was detected by RT-PCR and an overexpression of vIL-10 protein was found by SDS-PAGE analysis in pcDNA3.1（＋）/vIL-10 transfeted MSCs.Conclusion The vIL-10 mRNA and protein expressed in MSCs.