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OALib Journal期刊
ISSN: 2333-9721
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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
, PP. 623-627
Keywords: 腺病毒,胞嘧啶脱氨酶,基因治疗,血管内皮细胞生长因子,同源重组
Abstract:
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hindⅲ/bamhⅰ酶切位点的cd基因,亚克隆到prep8,继而用hindⅲ/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-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4.8×1012cfu/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。
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