应用AdEasy腺病毒载体系统构建人GST-π基因的重组腺病毒  

Construction of recombinant adenovirus plasmid containing GST-π gene

作　　者：罗小辑[1] 金先庆[1] 陈瑾[1] 

机构地区：[1]重庆医科大学附属儿童医院外科,重庆400014

出　　处：《重庆医科大学学报》2006年第4期466-469,493,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金重点项目(No.30330590)

摘　　要：目的:利用AdEasy腺病毒载体系统构建人谷胱甘肽转移酶-π(GST-π)基因重组腺病毒并在HEK293细胞中扩增制备重组病毒。方法:从质粒中通过PCR的方法扩增出目的基因GST-π并带有适宜的酶切位点,双酶切后连pAdtrackCMV中构建成腺病毒穿梭质粒pAdtrack-GST-π,经酶切线性化后,采用电穿孔转化到含腺病毒骨架质粒AdEasy1的BJ5183大肠杆菌电感受态细菌中,挑选同源重组质粒AdEasy-GST-π,酶切线性化重组质粒并转染HEK293细胞包装成重组病毒颗粒,荧光显微镜观察绿色荧光表达。重组病毒上清乒乓交互感染HEK293细胞,荧光显微镜观察绿色荧光表达。结果:经限制性内切酶检测和GFP表达证实成功地构建了携带GST-π基因的重组腺病毒载体并制备出高滴度重组病毒。结论:成功地构建了携带GST-π基因的重组腺病毒载体,为利用GST-π基因转染造血干细胞的基因治疗奠定了基础。Objective： To constuct recombinant adenovirus vector carrying the human glutathione transferase π （GST- π ）gene and amplify the adenovirus vector in HEK293cells. Methods： GST- π gene with the suitable enzyme site are. amplified from the original plasmid through PCR,then subcloncd into the shuttle plsmid pAdtrackCMV after digested by the same enzyme.The recombinant plasmid pAd- track-GST-π linearized by PmeI, plasmid pAdtrack-GST-π was electroporated into E. coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1. Picking up the homologous recombant plasmid AdEasy- GST-π ,then it was digested with PacI and transfected into HEK293 cells to package the adenovirus,followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscope. Recombinant virus supernatant infected HEK293 cells repeatedly,and the green fluorescence protein expression Was detected. Results： Through the restriction enzyme digestion and expression of GFP ,recombinant adenoviral vector GST- π gene was constructed successfully. Conclusions： The recombinant adenoviral vector containing GST-π gene was successfully constructed ,and based for the gene therapy of GST-π transferring the hematopoietic stem ceils.

关 键 词：腺病毒 GST-Π 基因工程 

分 类 号：R737.21[医药卫生—肿瘤]