人FRNK重组腺病毒的构建  

Construction of Human FRNK Recombinant Adenovirus Vector

作　　者：曹俊[1] 于皆平[1] 刘超红[2] 刘启胜[1] 齐元玲[1] 于红刚[1] 

机构地区：[1]武汉大学人民医院消化内科,湖北武汉430060 [2]中国科学院武汉病毒学研究所/病毒学国家重点实验室,湖北武汉430061

出　　处：《武汉大学学报（医学版）》2007年第3期265-269,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:30470782)

摘　　要：目的:应用AdEasy复制缺陷型腺病毒载体系统构建人FRNK基因重组腺病毒,并在HEK293细胞中扩增制备重组病毒。方法:把人FRNK基因克隆入腺病毒穿梭载体pAdTrack-CMV中,构建腺病毒质粒pAdTrack-CMV-hFRNK,经PmeⅠ内切酶酶切成线性化后,采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中,通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到HEK293细胞进行包装,获得人FRNK重组腺病毒pAdhFRNK。荧光显微镜下观察绿色荧光蛋白的表达。结果:经酶切和绿色荧光蛋白表达证明了hFRNK基因的重组腺病毒载体pAdhFRNK构建成功,并制备出高滴度的重组病毒。结论:成功构建携带人FRNK基因的重组腺病毒pAdhFRNK,为研究FRNK的基因治疗奠定了基础。Objective： To construct an hFRNK adenovirus vector, pAdhFRNK. Methods： Human FRNK was cloned into shuttle vector pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-hFRNK, and the resultant plasmid was linearized by Prne Ⅰ enzymatic digestion and was subsequently electroporated into E. coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1. Recombinants were screened by Kanamycin and were identified by observation of green fluorescent protein （GFP） expression and PCR. The Pac Ⅰ -linearized recombinant plasmid was transfected into HEK293 cells to package the adenovirus, and the hFRNK recombinant adenovirus was observed by fluorescent microscope. Results： The hFRNK recombinant adenoviral vector was constructed successfully, which was confirmed by restriction enzyme and GFP expression. Conclusion： The human FRNK recombinant adenovirus was constructed successfully, and provided a sound base for gene therapy.

关 键 词：FRNK 基因治疗 腺病毒 载体 

分 类 号：R730.264[医药卫生—肿瘤]