含血管内皮生长因子启动子驱动CD自杀基因重组腺病毒的制备及其体外杀伤作用观察

目的 应用AdEasier-1系统高效制备含血管内皮生长因子(VEGF)启动子驱动CD自杀基因重组腺病毒并观察其体外肿瘤杀伤作用。方法 以JM109细菌基因组为模板,PCR扩增出含Hind Ⅲ/Bam HⅠ酶切位点的CD基因,亚克隆到pREP8,继而用Hind Ⅲ/XbaⅠ切出含pA加尾信号的CD基因,插入到预先构建的含VEGF启动子的转移质粒pAdtrack-VEGFP构建pAdtrack-VEGFP-CD。经PmeⅠ线性化后转化AdEasier-1细菌,用25 μg/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-CD。经293细胞包装、扩增获得重组腺病毒Ad-VEGFP-CD,行PCR鉴定。通过测定细胞克隆形成率和细胞存活率观察重组腺病毒对体外培养的LoVo细胞的细胞毒作用。结果 构建重组腺病毒质粒的成功率为70%(7/10)。包装扩增后重组腺病毒的滴度为4.8×10¹² CFU/ml,PCR扩增结果与预期相符。在前药5-氟胞嘧啶存在下,重组腺病毒对LoVo具有明显杀伤作用,细胞克隆形成率和细胞存活率都明显下降。结论 应用AdEasier-1系统可以成功制备含VEGF启动子驱动CD自杀基因的重组腺病毒,该重组体具有明显的肿瘤杀伤作用。

Construction of recombinant adenoviruses containing cytosine deaminase gene driven by the vascular endothelial growth factor promoter using an AdEasier-1 system

Objective To efficiently construct a recombinant adenovirus containing cytosine deaminase (CD) gene driven by vascular endothelial growth factor (VEGF) promoter using an AdEasier-1 system and observe its killing effect on LoVo cells in vitro. Methods The CD gene was amplified by PCR, and amplicons were cloned in JM109 bacteria, and then recombined into pREP8 to obtain pREP8-CD, which was then digested by HindⅢand XbaⅠfor the CD fragment with polyadenylation site (CD-pA) subcloned into the VEGFP -containing shuttle plasmid pAdtrack-VEGFP to generate pAdtrack-VEGFP-CD-pA. After linearization with PmeⅠ, pAdtrack-VEGFP-CD-pA was transformed into AdEasier-1 cells, and the transformants were selected on LB agar plates containing 25 μg/ml kanamycin followed by identification of the positive pAdEasy-VEGFP-CD with electrophoretic analysis and enzymatic digestion. pAdEasy-VEGFP-CD was then digested with PacⅠand transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-CD, which was finally confirmed by PCR. The positive recombinant adenoviruses were transfected into LoVo cells to observe their in vitro anti-tumor effect. Results pAdEasy-VEGFP-CD was constructed with a success rate of 70%. After being packaged in 293 cells and purified by CsCl banding, the titer of the recombinant adenovirus Ad-VEGFP-CD reached as high as 4.8×10¹² CFU particle/ml, and the adenovirus was further confirmed by PCR analysis. In the presence of the prodrug 5-FC, the recombinant adenoviruses remarkably inhibited the growth of LoVo cells. Conclusion The recombinant adenoviruses containing CD gene under the control of VEGF promoter can be efficiently generated using the AdEasier-1 system, and exhibit potent anti-tumor effect in vitro.