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目的探讨构建携带人血管内皮细胞生长因子(hVEGF)的重组腺病毒载体,为进一步的基因转染构建组织工程骨的血管化提供实验基础。方法采用RT-PCR扩增的方法获得人源性的hVEGF165,并克隆入穿梭质粒pAdTrack CMV。构建的质粒pAdTrack CMV-VEGF165经酶切及测序鉴定正确后,通过pAdeasy1质粒的介导与腺病毒包装质粒pAdTrack CMV-VEGF165共转染至人胚肾细胞HEK293,经同源重组后获得携带人VEGF的重组腺病毒pAdTrack CMV-VEGF165。应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒,测定病毒滴度。结果 PCR鉴定证实重组腺病毒含有人VEGF,病毒滴度为0.5×1011pfu/ml。结论成功构建的携带人VEGF的重组腺病毒载体能在HEK293细胞内扩增获得足够高的病毒滴度,为基因治疗构建组织工程骨血管化的研究奠定基础。
Objective To investigate the construction of recombinant adenoviral vector carrying human vascular endothelial cell growth factor (hVEGF) and to provide experimental basis for further gene transfection into vascularization of tissue engineered bone. Methods Human hVEGF165 was obtained by RT-PCR amplification and cloned into the shuttle plasmid pAdTrack CMV. The recombinant plasmid pAdTrack CMV-VEGF165 was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pAdTrack CMV-VEGF165 was co-transfected into human embryonic kidney cell line HEK293 through pAdeasy1 plasmid and adenoviral packaging plasmid pAdTrack CMV-VEGF165. After homologous recombination, Of recombinant adenovirus pAdTrack CMV-VEGF165. The recombinant adenovirus was identified by PCR. The virus was purified from the plaque and purified by freeze-thaw cycles. The virus titer was determined. Results PCR identification confirmed the recombinant adenovirus containing human VEGF, virus titer of 0.5 × 1011pfu / ml. Conclusion The recombinant adenoviral vector carrying human VEGF can amplify the virus titer sufficiently in HEK293 cells and lay a foundation for gene therapy in the construction of tissue engineering bone vascularization.