摘要
目的包装及鉴定低氧反应元件(HRE)启动子控制下缺氧诱导目的基因hVEGF165基因表达的2型重组腺相关病毒。方法磷酸钙三质粒共转染方法将pAAV-HRE9-VEGF165、pAAV-Rep/cap、pHelper质粒转染HEK293T细胞,制备2型重组腺病毒相关病毒(rAAV2)。分离培养S-D大鼠心肌细胞,转染rAAV,细胞固定后做免疫细胞荧光染色,检测细胞内血管内皮细胞生长因子(VEGF165)蛋白的表达。结果pAAV-HRE9-VEGF165质粒经测序鉴定,结果与NCBI GenBank公布的人VEGF165cDNA核苷酸序列(AB021221)完全一致,含HRE启动子及目的基因hVEGF165的2型重组非复制型腺相关病毒包装成功,可感染原代培养的大鼠心肌细胞,缺氧可诱导VEGF165蛋白表达。结论成功包装并鉴定了rAAV-HRE9-hVEGF165载体,可有效转染心肌细胞,VEGF165基因的适时表达,为缺血性心肌疾病的“分子搭桥”治疗提供了更可能的安全性。
Objective To generate and identify an AAV - VEGF165 vector using a hypoxic response element promoter to control the expression of hVEGF165. Methods AVV vectors were prepared by using the three - plasmid cotransfection system. The AAV vector was cotransfected with two helper plasmids into HEK293T cells by calcium phosphate method, where one helper （pAAV- RC） mediates AAV vector replication, the other （pHelper） has AAV rep and cap genes. Myocardiocytes were isolated from neonatal mouse hearts and infected with AAV - HRE9 - VEGF165 vector. The expression of hVEGF165 protein was shown with immunofluorescence. Results The sequence result of plasmid AAV - HRE9 - VEGF165 was identical to that of human VEGF165 published by NCBI in GenBanK. The expression of hVEGF165 in myocardiocytes was elicited under anoxic condition, but not under normal non - anoxic condition. Conclusion An.adeno - associated virus vector （rAAV） was generated, containing a HRE （hypoxic response element promotor） and the objective gene VEGF165. The rAAV could infect myocardiocytes to transfer objective gene, and HRE9 could timely regulate the objective gene in response to the condition of myocardial ischemia.
出处
《徐州医学院学报》
CAS
2007年第2期71-74,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省教育厅重点资助项目(03JKA300140)
关键词
重组腺相关病毒
血管内皮细胞生长因子
低氧反应元件
recombine adeno -associated virus
vascular endothelial growth factor
hypoxic response element
