应用原位双膜法快速筛选人Flt3配体甲醇酵母高表达转化子  

In situ Two-film Method for Rapid Screening of Pichia pastoris Transformants Expressing High-level Human Flt3L

作　　者：张艳丽[1] 陈松森[1] 杨克恭[1] 狄旭[1] 熊安琪[1] 张友鸿[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院医学分子生物学国家重点实验室,北京100005

出　　处：《生物化学与生物物理进展》2002年第2期307-310,共4页Progress In Biochemistry and Biophysics

基　　金："十五"国家高技术"86 3"计划资助项目 ( 2 0 0 1AA2 12 171)~~

摘　　要：原位双膜法是一种基于免疫原理的快速筛选高表达甲醇酵母转化子的方法 ,即首先将固体培养基上的菌落转印至醋酸纤维素薄膜上 ,再利用硝酸纤维素薄膜原位捕获穿过醋酸纤维素薄膜的菌落外泌蛋白 ,然后用免疫方法检测与硝酸纤维素薄膜结合的蛋白 .利用此法筛选到人Flt3配体 (hFL)的甲醇酵母高表达转化子 ,液体诱导表达量约 2 0mg L .ELISA结果证明 ,原位双膜法所得的菌落染色强度与该菌落液体诱导表达水平正相关 .蛋白质印迹结果显示 ,培养上清在 2 5ku处有明显杂交条带 ,而对照组杂交呈阴性 ,且表达量随诱导天数增加 .原位双膜法是一种良好的筛选方法 ,可以快捷、准确地筛选高表达酵母转化子 .Pichia pastoris is a system to express a lot of foreign proteins, however the expression levels of transformants vary widely from one to one clone. In situ two-film method is a rapid method based on immunology to screen Pichia pastoris transformants expressing high-level protein, in which yeast colonies are lifted from solid culture onto a cellulose acetate film. Then a nitrocellulose film is used to capture the secreted proteins that pass through the cellulose acetate film. The proteins binding on the nitrocellulose film are stained by immunology methods. Pichia pastoris transformants expressing high-level hFL are abtained by using this method, and the expression level in liquid, culture is about 20 mg/L.. The result of ELISA shows that the staining intensity is correlated to the expression level in liquid culture. Western blot of culture supernatant shows obvious band at 25 ku, while the control doesn't show any band. The expression quantity increases as the inducing time increases.

关 键 词：原位双膜法 人Flt3配体 甲醇酵母 高表达转化子 表达 巴斯德毕赤酵母 

分 类 号：Q78[生物学—分子生物学]