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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。
Objective To construct human VEGF-C-siRNA adenovirus vector using AdMax adenovirus system and amplify 293 cells to prepare recombinant virus. Methods Specific siRNA targeting human VEGF-C mRNA was designed and synthesized. The corresponding double-stranded DNA was designed and synthesized, and cloned into pDC316-EG-FP-U6 vector to construct shuttle plasmid pDC316-VEGF-C siRNA-EGFP-U6 293 cells were cotransfected with the backbone plasmid pBHGF35 and packaged into recombinant virus particles. The green fluorescence was observed under a fluorescence microscope. Results The recombinant adenoviral vector carrying VEGF-C-siRNA was successfully constructed and verified by restriction endonuclease, PCR and GFP. Conclusion The recombinant adenovirus vector carrying VEGF-C-siRNA fragment was successfully constructed, which lays the foundation for the further study of anti-tumor lymphangiogenesis.