ki-67核心启动子调控E1A表达的腺病毒的构建和鉴定  

Construction and Identification of Adenoviruses with Ki-67 Core Promoter-regulated E1A Expression

作　　者：张俊文[1] 方胜 王佳琳 王佩文 张文欣 金贵善[1] 刘福生[1] ZHANG Junwen;FANG Sheng;WANG Jialin(Brain Tumor Research Center,Beijing Neurosurgical Institute,Beijing Tiantan Hospital,Capital Medical University,Beijing 100070,China)

机构地区：[1]北京市神经外科研究所脑肿瘤研究中心、首都医科大学附属北京天坛医院,100070

出　　处：《医学研究杂志》2024年第8期23-27,共5页Journal of Medical Research

基　　金：国家自然科学基金资助项目(52373129);北京市自然科学基金资助项目(7202020)。

摘　　要：目的构建含有ki-67启动子的腺病毒Ad-pki-67-E1A-GFP,实现特异在ki-67阳性的脑胶质瘤细胞中复制。方法利用分子生物学的方法将ki-67启动子序列克隆到pGL3-basic载体中,双荧光素酶报告基因实验检测ki-67启动子活性;将ki-67启动子构建到穿梭质粒,并与辅助质粒共转染293T细胞,重组腺病毒Ad-pki-67-E1A-GFP;Ad-pki-67-E1A-GFP加入到脑胶质瘤细胞中,荧光显微镜观察GFP表达情况,同时实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)法检测细胞中ki-67基因表达、腺病毒复制必需基因E1A表达和病毒拷贝数。结果克隆的ki-67序列在脑胶质瘤细胞中能够启动报告基因表达;酶切和测序证明ki-67启动子构建到穿梭质粒中,并重组得到腺病毒Ad-pki-67-E1A-GFP;Ad-pki-67-E1A-GFP能够感染脑胶质瘤细胞,并且E1A的表达和病毒拷贝数与细胞中ki-67表达呈正相关。结论ki-67启动子改造后的腺病毒在脑胶质瘤细胞中复制,为进一步改造用于基因治疗恶性脑胶质瘤奠定了基础。Objective To construct adenoviral Ad-pki-67-E1A-GFP containing the promoter of the ki-67gene to achieve replication specifically in ki-67-positive glioma cells.Methods The promoter sequence of the ki-67gene was cloned into the pGL3-basic vector using molecular biology methods.The ki-67 promoter activity was detected by the dual luciferase reporter gene assay.The ki-67 promoter was constructed into a shuttle plasmid and co-transfected with a helper plasmid in 293T cells to recombinant adenovirus Ad-pki-67-E1A-GFP.The Ad-pki-67-E1A-GFP was added to glioma cells,and the expression of GFP was observed by fluorescence microscopy.Meanwhile,the expression of ki-67gene,the expression of E1A gene,which was essential for adenovirus replication,and the number of virus copies were detected by real-time fluorescence quantitative polymerase chain reaction(PCR).Results The cloned ki-67sequence was able to activate reporter gene expression in glioma cells;enzymatic digestion and sequencing demonstrated that the ki-67 promoter was constructed into a shuttle plasmid and recombined to obtain the adenovirus Ad-pki-67-E1A-GFP.Ad-pki-67-E1A-GFP could infect glioma cells,and the expression of E1A and the number of virus copies were positively correlated with the expression of ki-67 in the cells.Conclusion The ki-67 promoter-modified adenovirus replicated in glioma cells,providing a basis for further modification for gene therapy of malignant gliomas.

关 键 词：ki-67启动子 腺病毒 脑胶质瘤 

分 类 号：R73[医药卫生—肿瘤]