猫多能性基因慢病毒表达载体的构建与鉴定  

Construction and identification of lentiviral vectors expressing cat pluripotency genes

作　　者：樊晓烨[1] 俞先峰[1] 袁宝[1] 陈健[1] 朱拓[1] 袁龙[1] 马云青[1] 栗楠[1] 谢珊姗 刘殿峰[1] 张嘉保[1] 任文陟[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062

出　　处：《扬州大学学报（农业与生命科学版）》2014年第1期13-15,26,共4页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31201866);高等学校博士学科点专项科研基金资助项目(20110061120100);吉林省科技发展计划项目(201101077)

摘　　要：从含有猫Oct4、Sox2、Klf4及c-Myc的质粒中获取目的基因,将目的基因与携带绿色荧光蛋白(GFP)的慢病毒载体LV5进行定向连接,构建慢病毒表达载体LV-Oct4、LV-Sox2、LV-Klf4、LV-c-Myc,将其连接产物分别转化至细菌感受态细胞中,经筛选阳性克隆、酶切鉴定后测序,通过lipofectamine2000介导转染293T细胞对慢病毒进行包装并测定病毒滴度,探讨猫多能性基因Oct4、Sox2、Klf4及c-Myc慢病毒载体的构建与包装。结果表明:经双酶切鉴定及测序分析,成功构建并包装出LV-Oct4、LV-Sox2、LV-Klf4及LV-c-Myc慢病毒载体,基因测序结果与目标序列完全一致,且病毒滴度均达到107 TU·mL-1以上。说明成功地构建了猫多能性基因慢病毒表达载体,获得稳定产生慢病毒颗粒的包装细胞株。The target genes were obtained from the plasmid containing cat Oct4, Sox2, Klf4 and c-Myc, and were directionally connected tothe lentiviral vector LV5 carrying green fluorescence protein （GFP） to construct the recombinant lentiviral vectors LV-Oct4, LV-Sox2, LV-Klf4 and LV-c-Myc. The product was respectively transformed into competent bacteria. The gene sequences were analyzed after identified by positive clones screening and restrictive enzyme digestion. Lipofectamine 2000 was used to transfect 293T cells for packing lentivirus and testing the titer of lentivirus. This paper aims to explore the construction and packing of lentivirus vectors including cat pluripotency genes Oct4, Sox2, Klf4 and c-Myc. The double enzyme digestion and DNA sequencing analysis revealed that the lentivirus vectors were suc- cessfully constructed and packed including Oct4, Sox2, Klf4 and c-Myc. The sequence of gene was consistent with objec- tive sequence and the titer of lentivirus reached over 107 TU·mL^-1. This study successfully constructed the lentiviral vectors expressing cat pluripotency genes and obtained the packaging cell lines stably producing lentiviral particles.

关 键 词：猫 多能性基因 慢病毒载体 胚胎干细胞 

分 类 号：R318[医药卫生—生物医学工程]