ADV-tk重组腺病毒的构建及检测  

Construction of Recombinant Adenovirus Containing tk Gene

作　　者：陈耿臻[1] 韩慧[1] 许铭炎[1] 邓小玲[1] 

机构地区：[1]汕头大学医学院第二附属医院外三科,广东汕头515041

出　　处：《中国普外基础与临床杂志》2010年第7期703-706,共4页Chinese Journal of Bases and Clinics In General Surgery

基　　金：广东省自然科学基金立项资助课题(项目编号:7301461);广东省科技计划项目立项资助课题(项目编号:2007B031514006);广东省医学科研基金立项资助课题(项目编号:B2008063)

摘　　要：目的应用基因重组技术,构建含胸苷激酶(tk)自杀基因的复制缺陷型腺病毒(ADV-tk),为进一步研究tk自杀基因对肿瘤的抑制作用奠定基础。方法采用细胞内同源重组法构建携带tk基因的ADV-tk,经PCR鉴定正确后进行扩增、纯化和滴度测定。重组腺病毒ADV-tk体外转染人肝癌SMMC-7721细胞,RT-PCR检测tk基因的整合和表达。建立裸鼠人肝癌动物模型,腹腔注射ADV-tk,荧光显微技术观察ADV-tk基因在肝脏的转染情况和对肿瘤细胞的抑制作用。结果构建的重组腺病毒中带有tk基因,经扩增、纯化后测得重组腺病毒滴度为1.4×1010pfu/ml。ADV-tk体外转染SMMC-7721细胞后,RT-PCR检测显示tk基因在细胞中整合并且有效表达。体内检测结果发现ADV-tk在肝脏有明显转染,肿瘤组织中凋亡细胞明显增多。结论本研究构建的携带tk基因的复制缺陷型腺病毒获得成功,可用于下一步研究。Objective To construct replication-defective adenovirus containing tk gene （ADV-tk）.MethodsRecombinant adenovirus of ADV-tk was constructed using homologous recombination in cells.After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR,the titre of purified recombinant adenovirus was detected.In vitro study,tk gene in SMMC-7721 cells transfected by ADV-tk was investigated by RT-PCR.In vivo study,ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed.Results Recombinant adenovirus containing ADV-tk was proved successfully.The titre of purified recombinant adenovirus was 1.4×10^10 pfu/ml.In vitro study,tk was integrated and expressed by SMMC-7721 cells.In vivo study,with the injection of ADV-tk,apoptosis cells in tumor increased.Conclusion A replication-defective adenovirus containing tk gene is successfully constructed,which may useful for further research on tumor suicide gene therapy with ADV-tk.

关 键 词：基因治疗 自杀基因 胸苷激酶基因 腺病毒载体 

分 类 号：R341[医药卫生—基础医学]