非洲爪蟾卵非细胞翻译体系的建立及应用  

Establishment and application of xenopus leavis egg cell-free translating system

作　　者：田辉凯[1] 姚小皓[1] 马兵[1] 张红梅[1] 李学军[1] 

机构地区：[1]北京大学基础医学院药理学系,北京100083

出　　处：《中国药理学通报》2003年第7期768-771,共4页Chinese Pharmacological Bulletin

摘　　要：目的　当前人类基因组研究的重心正在由“结构”向“功能”转移。而基因功能的研究离不开基因的表达 ,这就迫切需要一种操作简便 ,适用范围广的表达工具来表达有功能的蛋白质。方法　非洲爪蟾注射HCG催卵 ,2 %半胱氨酸去胶膜 ,高速离心制备卵提取物 ,即为非洲爪蟾卵非细胞翻译体系。通过亚克隆技术将TFAR1 9基因克隆到体外转录质粒中 ,通过酶切线性化模板 ,体外转录 ,加帽合成cRNA ,将其加入到制备的蛙卵提取物中进行体外翻译。结果　用Westernblot鉴定 ,获得了高效翻译 ,并且加入亚精胺和ATP再生系统可以提高翻译的效率。AIM To establish an efficient and convenient system for expressing functional proteins. METHODS Xenopus leavis females were injected with HCG, eggs were harvested next morning and dejellied by 2% cysteine, from which the Xenopus leavis egg cell free translating system was prepared. Through subcloning technique, TFAR19 gene was cloned to pBluescript SK plasmid which was linearized by enzyme, then transcript and capped in vitro to synthesis the cRNA of TFAR19. RESULTS This cRNA was translated in freshly prepared cell free translating system, the product was identified by Western Blot. CONCLUSION The results showed that this translating system could efficiently translate TFAR19 cRNA and be a very useful translating tool.

关 键 词：非细胞翻译体系 细胞凋亡 TFAR19 非洲爪蟾卵母细胞 

分 类 号：R-332[医药卫生] R282.71