血管内皮细胞生长因子165基因重组腺病毒的构建及其鉴定

Construction and identification of recombinant adenovirus containing human vascular endothelial growth factor 165 gene

目的:基因重组腺病毒具有较高的转染率,且宿主范围广。实验拟构建携带人血管内皮细胞生长因子165基因的重组腺病毒载体(Ad.VEGF165),为后续基因转染、微囊化基因工程细胞及其动物模型研究提供实验基础。方法:实验于2007-01/05在解放军第二军医大学长海医院胸心外科研究所实验室完成(国家级重点实验室)。①实验材料:pAxCAwt.VEGF165由长海医院胸心外科研究所惠赠。②实验方法:采用脂质体转染法,将pAxCAwt.VEGF165与DNA-TPC共转染人胚肾293细胞,扩增后获得载人血管内皮细胞生长因子165基因的复制缺陷型重组腺病毒。③实验评估:应用聚合酶链反应及酶切证实重组腺病毒中的目的基因。并根据50%组织培养感染剂量法计算病毒滴度。结果:①重组腺病毒Ad.VEGF165的构建:采用脂质体法可使pAxCAwt.VEGF165与DNA-TPC有效转染人胚肾293细胞,扩增出载人血管内皮细胞生长因子165基因的复制缺陷型重组腺病毒。②重组腺病毒Ad.VEGF165的鉴定:聚合酶链反应的产物进行NcoⅠ酶切鉴定,可得到597bp、146bp2个片段,与GeneTool软件理论上计算的结果完全一致,计算病毒滴度为2.2×1015pfu/L。结论:采用pAxCAwt.VEGF165与DNA-TPC系统经脂质体转染法成功构建的复制缺陷的重组腺病毒Ad.VEGF165滴度高、毒性低、效率高、体外转染安全。

AIM： Recombinant adenovirus possesses high transfection efficiency and wide host range. This study was designed to construct the recombinant adenovirus vector containing human vascular endothelial growth factor 165 （VEGF165）, so as to lay a foundation for the subsequent gene transfection, microencapsulated genetically engineered cells and animal experiments. METHODS： The experiment was conducted in the Laboratory of Cardiothoracic Surgery （the National Key Laboratory）, Changhai Hospital of The Second Military Medical University of Chinese PLA from January to May in 2007. Experiment materials： pAxCAwt.VEGF165 was provided by Institute of Cardiothoracic Surgery of Changhai Hospital. pAxCAwt.VEGF165 and DNA-TPC were cotransfected into human embryonic kidney 293 cells by lipofection method. Being propagated, recombinant replication-deficient adenovirus named Ad.VEGF165 was obtained. The target gene of recombinant adenovirus was identified by polymerase chain reaction （PCR） and restriction enzyme digestion. The titer of virus was detected by 50% tissue culture infective dose method. RESULTS： Construction of recombinant adenovirus Ad.VEGF165： The pAxCAwt.VEGF165 and DNA-TPC were successfully cotransfected into human embryonic kidney 293 cells by lipofection method, and replication-deficient adenovirus vectors coding for VEGF165 gene were generated. Identification of recombinant adenovirus Ad.VEGF165： Two fragments of PCR products （597 bp and 146 bp） were obtained by NcoⅠ restriction enzyme. The result was consistent with that calculated with Gene Tool software. The virus titers was 2.2×10^15 pfu/L. CONCLUSION： DNA-TPC and pAxCAwt.VEGF165 can be used to construct replication-deficient recombinant adenovirus Ad.VEGF165 in a high titer, low toxicity, high efficiency and safe transfection in vitro.