靶向人血管内皮细胞生长因子-C基因的siRNA腺病毒载体的构建

目的利用AdMax腺病毒系统构建人VEGF-C-siRNA腺病毒载体并在293细胞中扩增制备重组病毒。方法选择针对人VEGF-C mRNA的特异性siRNA靶序列,设计合成其相应的双链DNA,将其克隆入pDC316-EG-FP-U6载体中构建穿梭质粒pDC316-VEGF-C siRNA-EGFP-U6,再与骨架质粒pBHGF35共转染293细胞,包装成重组的病毒颗粒,荧光显微镜观察绿色荧光表达。结果经限制性内切酶、PCR检测和GFP表达证实成功地构建了携带VEGF-C-siRNA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VEGF-C-siRNA片段的重组腺病毒载体,为进一步抗肿瘤淋巴管生成的研究奠定了基础。

Construction of Recombinant Adenovirus Expressing the Small Interfering RNA of Vascular Endothelial Growth Factor-C

Objective To construct the adenovirus vector expressing the small interfering RNA of vascular endothelial growth factor-C by AdMax and amplify the adenovirus vector in 293 cells.Methods The specific siRNA sequence targeting human VEGF-C mRNA was selected.The homologous doublestrand DNA was designed and synthesized.After such DNA was inserted into pDC316-EGFP-U6 by screeening,pDC316-VEGF-C siRNA-EGFP-U6 was obtained,then it was co-transfected into 293 cells with the bone plasmid pBHGF35 to package the recombinant adenovirus,and the green fluorescence protein expression was detected.Results Recombinant adenoviral vector VEGF-C-siRNA was constructed successfully,which was confirmed by restriction cnzyme digestion,PCR and GFP expression.Conclusion The recombinant adenoviral vector carrying VEGF-C-siRNA is successfully constructed.and will lay a foundation of anti-lymphangiogenesis therapy for the colorectal cancer.