hHGF腺病毒载体的构建及其在人脐带间质干细胞中的表达

Transfection of human umbilical cord mesenchymal stem cells with recombinant adenovirus carrying human hepatocyte growth factor gene

目的:构建人重组肝细胞生长因子(human hepatocyte growth factor,hHGF)基因腺病毒载体(Ad-HGF),转染人脐带间质干细胞(human umbilical cord mesenchymal stem cells,hucMSCs),为转基因治疗提供实验基础。方法:设计含有SalⅠ及XbaⅠ酶切位点的引物,PCR扩增hHGF,将扩增产物克隆到带有绿色荧光蛋白(green fluorescenceprotein,GFP)标记的pAdTrack-CMV穿梭质粒上。重组穿梭质粒经PmeⅠ线性化后,与腺病毒骨架质粒pAdEasy-1在BJ5183中同源重组,筛选获得含有hHGF的重组腺病毒质粒,PCR、酶切鉴定并测序。重组病毒质粒用PacⅠ酶切线性化,脂质体转染293A细胞,经3轮扩增,制备高效表达的AdGFP/HGF,并转染hucMSCs。结果:重组腺病毒质粒经PCR和SalⅠ,XbaⅠ酶切鉴定,证实含有HGF基因,测序结果和设计片段的序列一致。荧光显微镜下发现293A细胞发光率几乎为100%,感染的hucMSCs亦可表达绿色荧光蛋白。结论:成功构建了hHGF腺病毒表达载体,并获得高滴度的病毒,能高效感染hucMSCs,为后续研究奠定了基础。

Objective： To construct green fluorescence protein（GFP） labeled recombinant adenovirus vector carrying human hepatocyte growth factor（hHGF） gene and transfect it into hucMSCs.Methods： The amplification products of HGF by PCR with a pair of primers with SalⅠ and XbaⅠ restriction endonuclease sites was subcloned into shuttle plasmid pAdtrack-CMV.The resultant plasmid,after linearized by digesting with restriction endonuclease PmeⅠ,was transformed into E.coli BJ5183 which had been transformed by adenoviral backbone plasmid pAdEasy-1.Recombinant plasmid was obtained and then confirmed by PCR and restriction endonuclease analysis.The adenovirus was packaged and propagated in human embryonal kidney cells（293A cells） after being linearized by digesting with restriction endonuclease PacⅠ.After three amplified,highly expression of AdGFP/HGF had been prepared,then transfect it into hucMSCs.Results： The results of PCR and restriction endonuclease assay indicated that target gene was inserted into recombinant adenovirus vector successfully.The sequence of fusion gene was the same as that of designed fragments.293A cells were found 100% luminance under fluorescence light microscopy.Modified hucMSCs could express GFP.Conclusion： Recombinant adenovirus vector containing hHGF has been constructed successfully and obtained highly efficient virus,and could transfect hucMSCs efficiently which laid a foundation for further investigation.