人低氧诱导因子-1α重组腺病毒的构建和鉴定

Construction and identification of recombinant adenovirus carrying human hypoxia-inducible factor-1α gene

目的利用AdMax腺病毒载体系统构建人重组低氧诱导因子-1α(rhHIF-1α)基因腺病毒并在293细胞中扩增制备重组病毒。方法自先期构建的pcDNA4-rhHIF-1α真核表达载体中用RT-PCR法扩增出rhHIF-1α基因片段,将其克隆入线性化的pEGFP1-C1质粒中,用PCR法扩增EGFP-rhHIF-1α基因片段,将其克隆入PUC18质粒中。酶切构建好的pUC18-rhHIF-1α载体并克隆进入穿梭质粒PDC316中,将构建好的穿梭质粒PDC316-rhHIF-1α载体和骨架病毒pBHGloxΔ1,3Cre共转染293细胞,包装成重组的病毒颗粒。重组的病毒转染SG-7901细胞,荧光显微镜观察绿色荧光表达。结果经限制性内切酶检测和GFP表达证实成功地构建了携带重组人HIF-1α基因的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带rhHIF-1α基因片段的重组腺病毒载体。

Objective To construct the adenovirus vector carrying the recombinant human hypoxia-inducible factor-1 and amplify the adenovirus vector in 293 cells. Methods The recombinant human HIF-1α gene was obtained from constructed plasmid pc-DNA4-rhHIF-1α by RT-PCR and then inserted into linearized pEGFP-C1 plasmid. The EGFP-rhHIF-1α gene segment was amplified by RT-PCR from pEGFP-rhHIF-1α vector and cloned into the plasmid PUC18. After having been screened, the constructed pUC18- EGFP-rhHIF-1α plasmid was digested with restriction endonueleases and cloned into the shuttle plasmid PDC316 to form PDC316- rhHIF-1α vector. The PDC316-rhHIF-1α plasmid was cotransfected with genomic plasmid pBHGlox△1,3Cre into 293 cells to package the recombinant adenovirus. The recombinant adenovirus was transfected into SG-7901 cells, and the green fluorescence protein expression was detected. Results Recombinant adenoviral vector Ad-rhHIF-1α was constructed successfully, and confirmed by restriction enzyme digestion and GFP expression. Conclusion The recombinant adenoviral vector carrying rhHIF-1α can be successfully constructed.