重组猪胰蛋白酶原及其突变体在毕赤酵母X33中的组成型表达  

Constitutive Expression of Recombinant Porcine Trypsinogen and its Mutant in Pichia pastoris X33

作　　者：窦俊 方宏清 徐登圆 赵珊珊 陈慧梅 徐晓峰 支艳艳 李晓稳 文良柱 DOU Jun;FANG Hong-Qing;XU Deng-Yuan;ZHAO Shan-Shan;CHEN Hui-Mei;XU Xiao-Feng;ZHI Yan-Yan;LI Xiao-Wen;WEN Liang-Zhu(China Pharmaceutical University,Nanjing 211198;Jiangsu Wanbang Pharmaceutical Technology Co.,Ltd.,Xuzhou 221004;Jiangsu Vonsun Pharmaceutical Technology Co.,Ltd.,Suzhou 215000,China)

机构地区：[1]中国药科大学,江苏南京211198 [2]江苏万邦医药科技有限公司,江苏徐州221004 [3]江苏万新医药科技(苏州)有限公司,江苏苏州215000

出　　处：《生物技术通讯》2020年第5期511-516,560,共7页Letters in Biotechnology

摘　　要：目的:设计以PGAP为启动子的组成型质粒,构建高效表达重组猪胰蛋白酶原的毕赤酵母X33菌株,获得重组猪胰蛋白酶。方法:首先将质粒转化大肠杆菌DH5α,提取质粒,电转化至毕赤酵母X33,表达重组猪胰蛋白酶原,经肠激酶激活,纯化提取得到重组猪胰蛋白酶。结果:筛选到高效表达重组猪胰蛋白酶原的毕赤酵母X33菌株,获得了重组猪胰蛋白酶。结论:以PGAP为启动子的质粒,转化毕赤酵母X33后表达的重组猪胰蛋白酶原避免了补加甲醇带来的危害,且操作方便,大大缩短了周期,提高了生产效率,避免了动物源性污染。Objective:To design a constitutive plasmid with PGAP as a promoter,construct a Pichia pastoris X33 strain that efficiently expresses recombinant porcine trypsinogen,and obtain recombinant porcine trypsin.Methods:The recombinant plasmids were transformed into E.coli DH5α.Then the plasmids were extracted and transformed into P.pastoris X33 to express recombinant porcine trypsinogen.The recombinant porcine trypsin was purified and extracted by enterokinase activation.Results:P.pastoris X33 was successfully screened for high expression of recombinant porcine trypsinogen,and we obtained recombinant porcine trypsin.Conclusion:The plasmid with PGAP promoter was transformed into P.pastoris X33 to express recombinant porcine trypsinogen,which could avoid the harm of methanol supplement,shorten the production cycle greatly,improve the production efficiency and avoid animal pollution.

关 键 词：毕赤酵母X33 重组猪胰蛋白酶原 重组猪胰蛋白酶 酵母表达 

分 类 号：Q78[生物学—分子生物学]