新法制备含VEGF启动子驱动的CD/TK融合基因重组腺病毒  

An easier method for generating recombinant adenovirus containing CD/TK fusi on gene driv-en by vascular endothelial growth factor promoter

作　　者：黄元媛[1] 黄宗海[1] 陈建发[1] 汤福祥[1] 杨文宇[1] 郑全[1] 车小燕[2] 

机构地区：[1]第一军医大学珠江医院普通外科,广东广州510282 [2]第一军医大学珠江医院中心实验室,广东广州510282

出　　处：《第一军医大学学报》2004年第1期117-120,共4页Journal of First Military Medical University

基　　金：国家"863"计划生物和现代农业技术研究基金(2001AA217171);广东省自然科学基金重点项目(013072)~~

摘　　要：目的 应用简化的细菌内同源重组法高效制备含血管内皮细胞生长因子(VEGF)启动子驱动的CD／TK融合基因重组腺病毒。方法 先以氯化钙法替代电穿孔法将腺病毒基因组质粒pAdEasy-1导入BJ5183细菌，制备BJ5183pAdEasy-1细菌；再以后者作感受态细菌，与线性化的转移质粒pAdtrack-VEGFP-CDglyTK进行同源重组，获得重组腺病毒质粒pAdEasy-VEGFP-CDglyTK，转染293细胞，制备含VEGF启动子驱动的CD／TK融合基因重组腺病毒。结果 构建重组腺病毒质粒的成功率为60％(6／10)。转染293细胞后7～12d可收获病毒。结论 简化的细菌内同源重组法简便易行，重组效率较高，易于推广应用。Objective To efficiently construct the recombinant adenov ir us containing CD/TK fusion gene driven by vascular endothelial growth factor (V EGF) promoter using improved homologous recombination in bacteria. Methods Chemi cal trans-formation, instead of electroporation, of the plasmid pAdEasy-1 into E.coli BJ5183 strain was performed to prepare BJ5183pAdEasy-1 as t he competent bacterium, in which pAdEasy-1 and pAdtrack-VEGFP-CDglyTK were recom bined. Fi-nally, the recombinant adenovirus of AdVEGFP-CDglyTK was constructed b y tranfecting 293 cells with linearized adenoviral genomes of pAdEasy-VEGFP-CDg lyTK. Results This new transformation procedure generated recombinant plasmids i n a yield of 60% (6/10), and the adenovirus AdVEGFP-CDglyTK was harvested 7-12 d after transfection. Conclusion The im-proved homologous recombination in bacte ria is efficient, convenient and easy to be carried out.

关 键 词：制备 VEGF 启动子 CD/TK融合基因 重组腺病毒 血管内皮细胞生长因子 基因治疗 肿瘤 

分 类 号：R73-3[医药卫生—肿瘤]