pcDNA3.1-bFGF真核表达载体的构建及其在犬骨髓基质干细胞中的表达

目的:研究碱性成纤维细胞生长因子(bFGF)真核表达载体转染犬骨髓基质干细胞(BMSCs)后的表达情况,并进一步研究bFGF基因转移在骨组织工程学中的应用。方法:提取国人脑胶质瘤细胞BT325总RNA,RT-PCR法扩增bFGF cDNA片段,构建重组载体pcDNA3.1-bFGF,重组载体经脂质体介导转染犬BMSCs,半定量RT-PCR及Western blot检测表达。结果:重组真核表达载体经PCR、酶切鉴定及测序证实正确。bFGF在犬BMSCs中获得表达。结论:成功构建了真核表达载体pcDNA3.1-bFGF,此重组载体在犬BMSCs中能够表达。

Construction of eukaryotic expression vector pcDNA3.1-bFGF and expression of the vector in canine bone marrow stem cells

Objective To explore the expression of eukaryotic expression vector bFGF in canine bone marrow stem cells(BMSCs) and to further study the application of bFGF gene transfer in bone tissue engineering.Methods Total cellular RNA of human glioma BT325 was isolated and RT-PCR amplification of the cDNA fragments of bFGF was performed.The fragment was cloned into vector pcDNA3.1.The eukaryotic expression vector was recombined and then transfected by lipofectamin into canine BMSCs.bFGF expression was analyzed using Western blot and RT-PCR.Results The recombinant vector pcDNA3.1-bFGF was identified by restriction enzyme analysis,PCR amplification and nucleotide sequencing.bFGF was successfully expressed in canine BMSCs.Conclusion The eukaryotic expression vector pcDNA3.1-bFGF,which can be expressed in canine BMSCs,has been successfully constructed.