表达绿色荧光蛋白和人类血管内皮生长因子的shRNA重组腺病毒的构建与鉴定

Construction and identification of recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor short hairpin RNA

目的构建表达绿色荧光蛋白（GFP）和人类血管内皮生长因子（VEGF）的重组腺病毒载体，并对其相关指标进行鉴定，观察其对人类鼻咽癌细胞系CNE的感染效果。方法使用基因工程技术将GFP和人类VEGF sRNA的cDNA通过LR法体外同源重组从质粒载体pGenesil-上转移至pGSadeno腺病毒表达载体上，得到腺病毒载体pGSadeno—GFP—VEGF，将PacI线性化的腺病毒DNA转染HEK293细胞，并在其中包装扩增腺病毒。采用PCR方法对重组腺病毒进行鉴定，利用穿梭质粒中所携带的GFP报告基因，进行病毒滴度的测定和对CNE细胞感染效率的检测。结果酶切鉴定及PCR结果证明GFP-EGF shRNA重组腺病毒载体构建成功，病毒滴度达到2×10^9PFU／ml，对CNE有较强的感染能力，感染腺病毒后CNE细胞的增殖速率明显下降。结论应用体外同源重组方法成功构建了表达GFP和VEGF shRNA的重组腺病毒载体。

Objective To construct and identify recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor （VEGF） iRNA and to inhibit the gene expression of VEGF in human nasopharyngeal carcinoma cell line CNE with the RNA interference （RNAi） technique, and explore the expression of VEGF in CNE and its proliferation before and after transfection. Methods By the method of restriction endonuclease digestion, the GFP and human VEGF genes were subcloned from pGenesil-1 plasmid into the shuttle vector pGSadeno-GFP-VEGF. After correct identification of the recombinant plasmid pGSadeno-GFP-VEGF, it was then digested by PacI and transfected into HEK293 cells to be packaged and amplification for rAd-VEGF. Meanwhile, the recombinant adenovirus was identified by PCR method. The expression of GFP was detected to evaluate the titre and transfective ratio. Results Digestion and PCR identification showed the construction of recombinant adenovirus rAd-VEGF was successful via LR recombinant. The titre was 2×10^9 PFU/ml while the transfective ratio in CNE cell line was more than 90 percent which met the need of experiment. Cell growth was inhibited compared with non-siRNA transfected CNE cell line. Conclusion The recombinant virus expressing GFP and VEGF are successfully constructed; it layed a foundation for further relevant study.