核基质结合区修饰的附着体载体介导的EGFP基因体外表达  

MAR-Modified Episomal Vector-Mediated EGFP Expression in Vitro

作　　者：梅文瀚[1] 吴兆平[1] 徐荣婷[1] 钱关祥[1] 卢健[1] 

机构地区：[1]上海交通大学基础医学院生物化学与分子生物学教研室,上海200025

出　　处：《上海交通大学学报（医学版）》2006年第11期1233-1238,共6页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(39970311);国家重点基础研究发展规划("973"计划)子课题基金(2004CB518804);上海市科委基金(04JC14041);上海交通大学基础医学院基金(2005JY10)资助项目

摘　　要：目的建立一个非病毒介导外源基因高效转移和稳定表达体系。方法采用EB病毒(EBV)来源的附着体质粒载体,并结合顺式作用元件IFN-MAR构建重组质粒pcDNA3-EGFP-EBVR和pcDNA3-EGFP-EBNA1-MAR。采用荧光显微镜观察并摄片,FACS及逆转录-聚合酶链反应检测EGFP表达阳性的COS-7细胞比例、荧光表达强度及EGFP基因的转录。通过Southern印迹和质粒还原实验分析质粒DNA在细胞中存在的形式。结果本实验构建的重组质粒载体pcDNA3-EGFP-EBVR和pcDNA3-EGFP-EBNA1-MAR在转染进入COS-7细胞后以染色体外附着体的形式存在。由于EBVR的存在,可以在体外获得EGFP较长时间的稳定表达。IFN-MAR替代O riP后,仍可在体外获得EGFP的稳定表达。结论MAR修饰的EBV附着体载体可以在体外获得外源基因高效稳定的表达。Objective To construct a sustained gene expression system mediated by nonviral vector. Methods Recombinant plasmid pcDNA-EGFP-EBVR which contains EGFP and EBVR （OriP and EBNA1 ） was constructed. Then Orip was replaced by matrix attachment region （MAR） from upstream of human β-IFN gene to construct pcDNA3-EGFP-EBNA1-MAR. Two plasmids were transfected into COS-7 cells and selected by G418. At different time points after transfection, EGFP expression was observed using fluorescent microscope photographs, FACS analysis and RT-PCR. At the same time, Hirt＇s DNA was extracted from the trasfected COS-7 cells, then existing pattern of plasmid DNA in COS-7 cells was analyzed by Southern blot and plasmid recovery. Results Compared with pcDNA3-EGFP, fluorescent cell percentage, EGFP expression and transcription level of pcDNA3-EGFP-EBVR and pcDNA3-EGFP-EBNA1-MAR-transfected COS-7 cells could both maintain stable in at least 60 d with G418 selection. The result of Southern blot and plasmid recovery indicated pcDNA3-EGFP-EBVR and pcDNA3-EGFP-EBNA1- MAR existed as episomal state in COS-7 cells. It was indicated that the application of EBVR could lead to long-term stable expression of exogenous gene in vitro, and IFN-MAR could substitute the function of OriP. Conclusion The application of EBVR and MAR can lead to long-term stable expression of exogenous gene.

关 键 词：基因治疗 EB病毒 复制子 附着体质粒载体 核基质结合区 

分 类 号：R346[医药卫生—基础医学]