细菌内重组法快速构建血管内皮生长因子165重组腺病毒载体

Construction of recombinant adenovirus vectors carrying vascular endothelial growth factor 165 with a method of homologous recombination in bacteria

背景:腺病毒载体具有在哺乳动物及其多种细胞基因转移和蛋白表达的高效性,目前以细菌内重组法构建病毒载体成为常用方法。目的:观察以细菌内重组法构建血管内皮生长因子165重组腺病毒载体的可行性。设计、时间及地点:基因转染病毒载体的单一样本实验,于2007-10/2008-02在中山大学分子生物实验室完成。材料:合成血管内皮生长因子165基因的引物合成和序列测定由上海生工公司提供。pDNR-1r质粒(已含有绿色荧光蛋白基因)、pInt.AV.spaT质粒、pInt.B.B质粒均购自广州拓普基因技术公司。方法:通过动态模板PCR基因合成法合成的血管内皮生长因子165基因克隆入pMD18-T载体获得pMD18-T-VEGF165。将pMD18-T-VEGF165和pDNR经EcoRⅠ和BamHⅠ双酶切,构建pDNR-VEGF165载体,酶切鉴定后,pDNR-VEGF165与pInt.AV1.spat共转化BNN菌株的感受态细胞,构建pInt.AV1.spat.VEGF165腺病毒表达载体,将线性化的pInt.AV1.SpaT-VEGF165和pInt.B.B转染293细胞。主要观察指标:以DNA测序、酶切及聚合酶链反应法鉴定重组质粒和病毒,并测定重组腺病毒的感染效率。结果:重组质粒用EcoRⅠ酶切后,切成2条片段,大小约为1kb和6.9kb,实际的酶切分析结果与理论分析相符,证明转化子中的重组质粒为Cre-LoxP系统介导的含有血管内皮生长因子165基因的重组质粒。荧光显微镜下,8h后即可见部分细胞发出荧光,24h发荧光的细胞数及强度达到高峰,转染率超过80%。线性化的pInt.AV1.SpaT-VEGF165和pInt.B.B共转染293细胞,12～14d后会出现细胞病变效应。分别提取病变293细胞、正常293细胞的DNA,PCR后,1%琼脂糖凝胶电泳,病变的293细胞在约500bp处出现条带。结论:以细菌内重组法获得了含有成熟血管内皮生长因子165基因的重组腺病毒。

BACKGROUND： Adenovirus vectors have a highly efficient gene transfer and protein expression in mammals and many kinds of their cells. At present, viral vectors are normally constructed with the method of homologous recombination in bacteria. OBJECTIVE： To explore the feasibility of constructing recombinant adenovirus vector carrying vascular endothelial growth factor 165 （VEGF165） with the method of homologous recombination in bacteria. DESIGN, TIME AND SETTING： Single sample experiment of gene transfection viral vector was performed at Laboratory of Molecular Biology, Sun Yat-sen University from October 2007 to February 2008. MATERIALS： Primer synthesis and sequencing of VEGF165 were conducted by Shanghai Sangon Biological Engineering Technology ＆ Services Co., Ltd. Plasmid pDNR-1r （containing green fluorescent protein gene）, pInt.AV.spaT and pInt.B.B were bought from Guangzhou Topo Gene Technology Co., Ltd. METHODS： The pMD18-T-VEGF165 was obtained by cloning VEGF165 synthesized with dynamic template PCR gene synthesis method into pMD18-T vector. Then pMD18-T-VEGF165 and pDNR-Ir was digested by EcoR Ⅰ and BamHⅠ to construct pDNR-VEGF165 vector, after which pDNR-VEGF165 and pInt.AV1.spat were used in the cotransformation of competent cells of BNN strain to construct pInt.AV1.spat.VEGF165 adenovirus expression vector. At last, the linearized pInt.AV1.SpaT-VEGF165 and pInt.B.B were transfected into 293 cells. MAIN OUTCOME MEASURES： Sequencing, enzyme digestion and PCR were used to detect recombinant plasmid and adenovirus, and to evaluate the infection efficiency of Ad-VEGF165. RESULTS： After being digested with EcoRⅠ into two fragments of 6.9 kb and 1 kb, the recombinant plasmid presented the same enzyme digestion analysis results as theoretical analysis results, which showed that the combinant plasmids in the converter were the Cre-LoxP-mediated ones carrying VEGF165. Under fluorescence microscope, parts of cells could be seen to emit fluorescence after 8 hours following digestion, and the fluorescigenic cells reached a peak at hour 24 with a transfection efficiency over 80%. The 293 cells cotransfected with pInt.AV1.SpaT-VEGF165 and pInt.B.B showed cytopathogenic effects at 12-14 days after infection. Straps appeared at about 500 bp of the pathological 293 cells, according to the 1% agarose gel electrophoresis taken to the DNA of both the normal 293 cells and pathological ones after PCR. CONCLUSION： Recombinant adenovirus vectors carrying mature VEGF165 genes were successfully constructed with the method of homologous recombination in bacteria.