重组人血管内皮生长因子165腺病毒载体的构建与鉴定

Construction and identification of recombinant human vascular endothelial growth factor 165 gene adenoviral vector

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 construct adenovirus vector of the human vascular endothelial growth factor （hVEGF）, for further gene transfer to provide an experimental basis of constructing tissue engineering bone of vascularization. Methods Amplified hVEGF-165 by RT-PCR method,and cloned it into the pAdTrack CMV shuttle plasmid. To identification sequencing of the plasmids of pAdTrack CMV-VEGF165 by restriction enzyme digestion. By pAdeasy 1 plasmid mediated and adenovirus packaging plasmid of pAdTrack CMV-VEGF165 to transfect into human embryonic kidney cells HEK293 ,to obtain recombinant adenovirus carrying human VEGF pAdTrack CMV-VEGF-165 by homologous recombination. Identification of adenovirus by PCR, Plaque-purified virus passaged, amplified virus through repeated freezing and thawing, to determine the virus titers. Results PCR confirmed that recombinant adenovirus containing the human VEGF gene, virus titer was 0. 5 ×10^ll pfu/ml. Conclusions Successfully constructed recombinant adenovirus vector carrying human VEGF and can be amplified sufficiently high viral titers in HEK293 cells, to lay the foundation study for gene therapy of tissue engineering bone of vascularization.