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目的:构建携带人血管内皮生长因子(VEGF)165基因的重组腺病毒载体。方法:应用EcoRⅠ和XbaⅠ酶切质粒PDC-VEGF和PDC315,连接DNA目的片段后转化大肠杆菌DH5α构建重组质粒PDC315-VEGF,对重组质粒进行酶切分析和测序鉴定。通过脂质体将质粒PDC315-VEGF与质粒pBHGE3共转染293细胞获取重组腺病毒,以重组腺病毒DNA为模板,PCR鉴定构建的重组腺病毒。扩增、浓缩纯化重组腺病毒,微量滴定法测定腺病毒滴度。结果:通过连接反应构建重组质粒PDC315-VEGF,酶切分析和测序证明构建正确。经细胞内同源重组构建携带人VEGF165基因的重组腺病毒,PCR扩增421bpVEGF165片段,证实VEGF165基因成功克隆到复制缺陷型腺病毒载体,腺病毒滴度为5.0×109pfu/mL。结论:通过双质粒细胞同源重组,生产携带人VEGF165基因的重组腺病毒,为动物试验奠定基础。
OBJECTIVE: To construct recombinant adenoviral vector carrying 165 gene of human vascular endothelial growth factor (VEGF). Methods: The plasmids PDC-VEGF and PDC315 were digested with EcoRⅠ and XbaⅠ. After the DNA fragment was ligated, the recombinant plasmid was transformed into E. coli DH5α to construct recombinant plasmid PDC315-VEGF. The recombinant plasmids were digested and sequenced. Recombinant adenovirus was obtained by cotransfection of plasmid PDC315-VEGF and plasmid pBHGE3 into 293 cells by liposome, and the recombinant adenovirus was identified by PCR using recombinant adenovirus DNA as a template. Amplification, concentration and purification of recombinant adenovirus, microtiter method for the determination of adenovirus titer. Results: The recombinant plasmid PDC315-VEGF was constructed by ligation reaction, and digestion analysis and sequencing proved that it was correctly constructed. Recombinant adenoviruses harboring human VEGF165 gene were constructed by intracellular homologous recombination, and 421bp VEGF165 fragment was amplified by PCR. The results showed that VEGF165 gene was successfully cloned into replication-deficient adenovirus vector. The titer of adenovirus was 5.0 × 109pfu / mL. Conclusion: Recombinant adenovirus carrying human VEGF165 gene can be produced by homologous recombination of two plasmids, which lays the foundation for animal experiments.