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背景:血管内皮生长因子能促进内皮细胞分裂增殖及血运重建,诱导血管生成,近年来以血管内皮生长因子为基础的基因治疗已逐步试用于临床。但质粒载体转染率低、腺病毒载体对机体的免疫原性及存在潜在感染危险等问题尚需探讨。目的:拟构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒,鉴定并测定病毒滴度及活性。设计、时间和地点:开放性实验,于2007-03/09在陕西省疾病预防控制中心病毒室完成。材料:病毒包装细胞AAV-293、病毒滴度检测细胞AAV-HT1080购自Stratagene公司;EcoliDH5α菌株由陕西省疾病预防控制中心保存;AAVhelper-freesystem腺相关病毒包装系统中pAAV-IRES-hrGFP质粒包含绿色荧光报告基因,购自Stratagene公司;重组携带hVEGF165基因的质粒pUC18-hVEGF165由西安交通大学第二医院骨科时志斌博士构建。方法:从含有hVEGF165基因的pUC18-hVEGF165质粒中扩增出hVEGF165片段,插入腺相关病毒骨架质粒pAAV-IRES-hrGFP,构建重组骨架质粒pAAV-hVEGF165-IRES-hrGFP。将此重组质粒和腺相关病毒包装质粒pAAV-RC、辅助质粒pAAV-Helper磷酸钙法共转染AAV-293细胞,通过同源重组产生重组腺相关病毒rAAV-hVEGF165-GFP。荧光显微镜下监测病毒包装效率,裂解AAV-293细胞收获重组病毒并进行浓缩纯化。应用该重组病毒感染AAV-HT1080细胞,荧光计数法测定病毒滴度,并通过病毒基因组外源基因hVEGF165扩增鉴定重组病毒的包装是否成功。应用该重组病毒感染AAV-HT1080细胞。主要观察指标:荧光显微镜下监测病毒包装效率。荧光计数法测定病毒滴度,并通过病毒基因组外源基因hVEGF165扩增鉴定重组病毒的包装是否成功。结果:扩增产物经DNA测序确定为hVEGF165cDNA片段,重组骨架质粒pAAV-hVEGF165-IRES-hrGFP经双酶切鉴定正确。病毒包装效率达95%以上,收获纯化病毒滴度达5.5×1011vg/mL。提取重组病毒基因组成功扩增出外源目的基因片段hVEGF165,证实重组病毒rAAV-VEGF165-GFP包装成功。结论:成功构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒rAAV-VEGF165-GFP,收获的病毒具有较高滴度和感染活性。
BACKGROUND: Vascular endothelial growth factor (VEGF) can promote endothelial cell division, proliferation and revascularization and induce angiogenesis. In recent years, gene therapy based on vascular endothelial growth factor has been gradually applied to clinical trials. However, low vector plasmid transfection, adenovirus vector on the body’s immunogenicity and the potential risk of infection and other issues still need to be explored. OBJECTIVE: To construct a non-pathogenic recombinant adeno-associated virus with green fluorescent protein (EGFP) tag and carrying hVEGF165 gene, and to identify and measure the virus titer and activity. DESIGN, TIME AND SETTING: The open experiment was performed in the virus room of Shaanxi Provincial Center for Disease Control and Prevention on March 03, 2007. Materials: Virus packaging cell AAV-293, virus titer detection cells AAV-HT1080 were purchased from Stratagene; Ecoli DH5α strain was deposited by Shaanxi Provincial Center for Disease Control and Prevention; pAAV-IRES-hrGFP plasmid in AAVhelper-freesystem adeno-associated virus packaging system contained green Fluorescent reporter gene was purchased from Stratagene company; recombinant plasmid pUC18-hVEGF165 carrying hVEGF165 gene was constructed by Dr. Shi Zhibin, Orthopedics, Second Hospital, Xi’an Jiaotong University. Methods: hVEGF165 fragment was amplified from pUC18-hVEGF165 plasmid containing hVEGF165 gene and inserted into adeno-associated virus backbone plasmid pAAV-IRES-hrGFP to construct recombinant backbone plasmid pAAV-hVEGF165-IRES-hrGFP. Recombinant adeno-associated virus rAAV-hVEGF165-GFP was generated by homologous recombination of the recombinant plasmid and the adeno-associated virus packaging plasmid pAAV-RC and the helper plasmid pAAV-Helper calcium phosphate. The virus packaging efficiency was monitored under a fluorescence microscope, the recombinant virus was harvested from AAV-293 cells and concentrated and purified. The recombinant virus was used to infect AAV-HT1080 cells. Fluorescent counting was used to determine the virus titer. The recombinant virus was packaged successfully by hVEGF165 amplification of the viral genome. The recombinant virus was used to infect AAV-HT1080 cells. MAIN OUTCOME MEASURES: Virus packaging efficiency was monitored under a fluorescence microscope. Fluorescent counting method was used to determine the titer of the virus and the packaging of the recombinant virus was identified by the hVEGF165 amplification of the viral genome. Results: The amplified product was identified as hVEGF165 cDNA by DNA sequencing. The recombinant plasmid pAAV-hVEGF165-IRES-hrGFP was identified by double enzyme digestion. Virus packaging efficiency of more than 95%, harvesting purified virus titer of 5.5 × 1011vg / mL. The hVEGF165 exogenous gene fragment was successfully amplified by extracting the recombinant virus genome and the packaging of rAAV-VEGF165-GFP recombinant virus was confirmed. Conclusion: The recombinant adeno-associated virus rAAV-VEGF165-GFP with green fluorescent protein (EGFP) tagged with hVEGF165 gene was constructed successfully. The harvested virus has high titer and infectivity.