突变型hIL-2在Pichia pastoris中直接诱导表达条件研究  

Studies on the Conditions of the Direct Induce Expression of Mutant Variant Human Interleukin-2 in Pichia pastoris

作　　者：郭芝刚[1] 欧阳克清[1] 杨红[1] 高虹[1] 李新平[1] 陈云高 蔡绍皙[1] 

机构地区：[1]重庆大学分子生物学重点实验,重庆400044 [2]成都荣高实业集团有限公司,四川成都610000

出　　处：《生物技术》2003年第3期11-13,共3页Biotechnology

摘　　要：为了简化突变型白细胞介素 - 2 (mutantvarianthumaninterleukin - 2 ,MvIL - 2 )在毕赤酵母中的表达工艺 ,作者参照传统方法 ,设计了一种新的表达方法 -直接诱导法。在该实验中 ,在摇瓶条件下 ,对OD6 0 0 、诱导时间这两个重要的参数进行了重点研究 ,并对表达蛋白的抗原性和生物学活性进行鉴定。结果表明 :在pH6 0、温度 30℃、转速 2 80r min的条件下进行培养 ,菌体密度(OD6 0 0 )达到 4 0时直接在发酵液中加入 1%的甲醇进行诱导表达 ,间隔 2 4h补充同上浓度的甲醇 ,96h收集发酵液。以相同体积的培养基最后获得的总目的蛋白来比较 ,直接诱导法的产量是传统诱导法产量的 5倍。Westernblot的抗原性检测和MTT生物学活性实验证明表达的蛋白是特异性蛋白。MTT法测得突变型IL - 2具有 4倍于同样在毕赤酵母中表达的天然IL -In order to simplify the expressing technical process of mutant variant human interleukin-2 in pichia pastoris,we introduce a kind of new expressing method,directly inducing approach,referring to the traditional method In the experiment,the two parameters,OD 600 and inducing time,have been observed carefully when shaking bottles,then we appraised the speciality and biological activity of the MvIL-2 On the conditions of pH 6 0,30℃,rotating speed 280r/min and OD 600 40,we add 1% methanol to induce expressing protein,supplement the same methanol every 24 hours then collect the supernatants after 96 hours Comparing the total objective protein yielded in the two methods in the same culture medium,we find that the output of the directly inducing method is 5 times more than that of the traditional method The protein is tested to be objective protein by Western blot and MTT,and the biological activity of this MvIL-2 is 4 times of the NhIL-2 by MTT

关 键 词：突变型hIL-2 诱导表达 突变型白细胞介素-2 毕赤酵母 

分 类 号：Q786[生物学—分子生物学]