摘要
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 a recombinant adenovirus containing the human vascular endothelial growth factor 165 （VEGFl65）gene. Methods Plasmid pDC315 and plasmid pDC-VEGF were digested with EcoR Ⅰ and Xba Ⅰ , ligated a target fragment of DNA, then transformed into DH5α competent cells to construct recombinant plasmid pDC315- VEGF. The plasmid was analyzed with restriction enzyme digestion and identified by sequencing. The identified plasmid was then cotransfected with pBHGE3 into 293 cells to generate a recombinant adenovirus. The constructed adenovirus was identified by PCR and then amplified and purified. PFU titers were determined by plaque assays. Results Plasmid pDC315-VEGF was identified by restriction enzyme analysis and DNA sequencing to have a full length of human VEGF165 cDNA fragment. The adenovirus vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR, with a virus concentration of 5.0 × 10^9 pfu/mL. Conclusion A recombinant adenovirus vector containing human VEGF165 gene has been successfully constructed via homologous recombination with double plasmids.
出处
《实用医学杂志》
CAS
北大核心
2009年第3期342-344,共3页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(编号:30470701
30470702
30570747
30670873)
国家973项目(编号:2006CB503803)
