DD3启动子调控TRAIL过表达载体的构建及腺病毒包装  

Construction and adenovirus packaging of DD3 promoter regulated TRAIL over expression vector

作　　者：刘雷[1] 黄星华[1] 李坚伟[1] 周建华[1] 陈统权 

机构地区：[1]深圳市龙岗区人民医院,广东深圳518172

出　　处：《现代肿瘤医学》2016年第17期2686-2689,共4页Journal of Modern Oncology

基　　金：深圳市龙岗区科技计划资助项目(编号:YS2013024)

摘　　要：目的:构建DD3(PCA3)启动子调控的肿瘤坏死因子相关凋亡诱导配体TRAIL过表达腺病毒载体,并对其进行病毒包装及滴度测定。方法:将DD3启动子用分子克隆技术替换掉穿梭质粒p HBAd-U6-GFP原有启动子U6,再用Age I单酶切将制备的重组质粒p HBAd-DD3-GFP线性化,将TRAIL基因片段克隆至线性化p HBAd-DD3-GFP上,经抗性筛选后PCR及测序鉴定为p HBAd-DD3-TRAIL载体。将其连同病毒骨架质粒p HBAd-BHG共同转染至HEK293细胞包装成病毒并扩增,测定病毒感染性滴度。结果:经PCR及测序验证证实成功构建DD3启动子调控的肿瘤坏死因子相关凋亡诱导配体TRAIL过表达腺病毒载体p HBAd-DD3-TRAIL并包装扩增,病毒滴度为1.58×1010PFU/ml。结论:构建DD3启动子调控的凋亡配体TRAIL过表达重组腺病毒,可用于下一步在前列腺癌细胞中特异性地表达及诱导细胞凋亡杀伤靶细胞效应研究中。Objective：To construct the TNF-related apoptosis inducing ligand（ TRAIL） gene adenovirus expression vector modulated by DD3（ PCA3） promoter,and to proceed with the adenovirus package and titer determination.Methods：DD3（ PCA3） promoter cloned into p HBAd-U6-GFP shuttle plasmid to replace U6.p HBAd-DD3-GFP was linearized with Age I.TRAIL gene was inserted into p HBAd-DD3-GFP.The positive clones were identified by PCR,and analyzed by sequencing.p HBAd-DD3-TRAIL and framework plasmid p HBAd-BHG were co-transfected into HEK293 cells to package the adenovirus.The title of the lentivirus was measured.Results：PCR and sequencing that the TNF-related apoptosis inducing ligand（ TRAIL） gene adenovirus expression vector modulated by DD3（ PCA3） promoter had been successfully constructed.The titer of the virus was 1.58 × 10^10 PFU / ml.Conclusion：The TNF-related apoptosis inducing ligand（ TRAIL） gene adenovirus expression vector modulated by DD3（ PCA3） promoter is successfully constructed,and this lays the foundation for the research of specifically induce death of prostate cancer.

关 键 词：腺病毒 TRAIL DD3 PCA3 

分 类 号：R730.51[医药卫生—肿瘤]