应用AdEasy腺病毒载体系统构建血管内皮生长因子165重组腺病毒

Construction of Recombinant Adenovirus Vector Carrying the Vascular Endothelial Growth Factor 165 Using the AdEasy System

目的利用AdEasy腺病毒载体系统构建血管内皮生长因子165（VEGF165）重组腺病毒，并在293T细胞中扩增。方法将PCR获取的VEGF165基因酶切并插入到pAdTrack-CMV中，构建成腺病毒穿梭质粒pAdTrack-VEGF165，经PmeI酶切线性化后，采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌感受态细胞中，挑选同源重组菌落。提取质粒并用Pac I酶切鉴定，线性化重组质粒pAdEasy-VEGF165转染293T细胞，包装成重组腺病毒颗粒，荧光显微镜下观察绿色荧光表达，并扩增收集重组腺病毒，测定病毒滴度。结果经限制性内切酶检测、基因测序及和绿色荧光观察证实成功构建了携带VEGF165基因的重组腺病毒，并扩增出10^9pfu／mL的高滴度重组腺病毒。结论成功构建了携带VEGF165基因的重组腺病毒载体，为研究骨组织工程血管化局部基因治疗奠定了基础。

Objective To construct recombinant adenovirus vector carrying vascular endothelial growth factor 165 （VEGF165） and amplify the adenovirus vector in 293T cells. Methods VEGF165 obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdTrack-CMV to generate recombinant plasmid pAdTrack-VEGF165, and then the Pme I-linearized plasmid pAdTrack-VEGF165 was eleetroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1. The identified recombinant plasmid pAdEasy-VEGF165 DNA was digested with Pac I and transfered into 293T cells to package adenovirus, followed by identification of the recombinant adenovirus by means of observation of the enhanced green fluorescence protein （EGFP） expression under fluorescent microscope. After amplified in 293T cells, the obtained adenovirus was transfered into 293T cells again, and EGFP expression was detected. Results Recombinant adenoviral VEGF165 was constructed successfully and confirmed by restriction enzyme digestion, gene sequence and EGFP expression. Conclusion The recombinant adenoviral vector carrying VEGF165 was successfully constructed, and provided the basis of VEGF165 gene therapy.