应用AdEasier-1系统高效制备含VEGFP驱动TK自杀基因的重组腺病毒  

Construction of Recombinant Adenoviruses Encoding TK Suicide Gene Driven by VEGF Promoter Using Efficient AdEasier-1 System

作　　者：陈建发[1] 黄宗海[1] 黄元媛[1] 宋慧娟[1] 车小燕[2] 

机构地区：[1]第一军医大学珠江医院普外科,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：《癌症》2004年第9期1093-1097,共5页Chinese Journal of Cancer

基　　金：国家"863"项目(No.2001AA217171);广东省自然科学基金重点项目(No.013072)~~

摘　　要：背景及目的:自杀基因疗法的瓶颈之一是自杀基因在肿瘤细胞的特异性表达水平低。利用肿瘤特异性启动子来调控自杀基因使其在肿瘤细胞中特异性表达已成为肿瘤自杀基因研究的热点。研究证实血管内皮生长因子在绝大多数实体瘤细胞中都有过量表达而在正常组织中不表达或表达甚微,其过量表达与血管内皮生长因子启动子(vascularendothelialgrowthfactorpromoter,VEGFP)活性上调有关。为研究VEGFP可否提高TK自杀基因在肿瘤细胞的特异性表达水平,本研究应用一种高效的重组腺病毒构建系统,即AdEasier-1系统制备含VEGFP驱动TK自杀基因的重组腺病毒。方法:VEGFP自pEGFP-1-SV-VEGFP切下后插入pAdtrack载体上构建pAdtrack-VEGFP。用HindⅢ/XbaⅠ自pREP8-TK切出带polyA加尾信号的TK基因,亚克隆到pAdtrack-VEGFP中构建转移质粒pAdtrack-VEGFP-TK。PmeⅠ酶线性化转移质粒pAdtrack-VEGFP-TK,转化含腺病毒基因组质粒pAdEasy-1的细菌AdEasier-1,用25μg/ml卡那霉素筛选阳性克隆,先后进行琼脂糖电泳和PacⅠ酶切鉴定,筛选出正确重组腺病毒质粒pAdEasy-VEGFP-TK,经293细胞包装、扩增获得重组腺病毒Ad-VEGFP-TK,行酶切鉴定、PCR鉴定和测序。结果:卡那霉素抗性细菌只有两种,一种含pAdEasy-VEGFP-TK(大于33kb)。BACKGROUND &OBJECTIVE: One of the bottlenecks of suicide gene th er apy is the low specific expression of suicide genes in tumor cells. Using tumor specific promoter to modulate suicide genes resulting in high specific expressio n of genes in tumor cells has became a hot research topic of tumor suicide gene therapy. It has showed that vascular endothelial growth factor (VEGF) over-expr esses in almost all solid tumors, but not in normal tissues, which is highly rel evant to the up-regulation of VEGF promoter (VEGFP) activity. This study was to use the simplified and efficient AdEasier-1 system to gener ate recombinant adenoviruses encoding TK gene driven by VEGFP, and determine whe ther VEGFP could increase the expression of TK suicide gene in tumor cells. METH ODS:A fragment containing VEGFP was isolated from pEGFP-1-SV-VEGFP (Not Ⅰ/Xh o Ⅰ), and cloned into the shuttle plasmid pAdTrack resulting in pAdTrack-VEGFP . TK gene by polyadenylation site was cut from pREP8-TK by HindⅢ,and XbaⅠ, an d subcloned into pAdTrack-VEGFP resulting in pAdtrack-VEGFP-TK, which was lin earized by PmeⅠand transformed into AdEasier-1 Cells. Transformants were selec ted on LB agar plates containing 25 ìg/mL kanamycin, and positive pAdEasy-VEGF P-TK was identified by electrophoretic analysis and enzymatic digestion, then d igested with PacⅠ, and transfected into 293 cells to produce the recombinant ad enovirus Ad-VEGFP-TK. Ad-VEGFP-TK was finally confirmed by polymerase chain reaction (PCR) procedure, and DNA sequence analysis. RESULTS: The pAdTrack-VEGF P-TK (about 12 kb)and pAdEasy-VEGFP-TK(larger than 33 kb), both selectable by kanamycin resistance, could be clearly identified by electrophoretic analysis . Digesting pAdEasy-VEGFP-TK with PacⅠresulted in 1 specific small fragment ( about 3.0 kb), and 1 large fragment (larger than 33 kb). The pAdEasy-VEGFP-TK was successfully constructed at a frequency of 60%(6/10). After being packaged in 293 cells, and purified by CsCl banding, the recombinant adenovirus Ad-VEGFP-TK,

关 键 词：肿瘤 重组腺病毒 载体 自杀基因治疗 血管内皮生长因子 

分 类 号：R73-3[医药卫生—肿瘤]