新型毕赤酵母分泌表达载体的构建及功能验证.pdfVIP

第40 卷第7 期 东 北 农 业 大 学 学 报 40(7): 55~59 2009 年7 月 Journal of Northeast Agricultural University July 2009 新型毕赤酵母分泌表达载体的构建与功能验证 宋庆凤，暴立娟，李 杰* （东北农业大学生命科学学院，哈尔滨 150030） 摘 要：巴斯德毕赤酵母（Pichia pastoris）是目前应用最广泛的外源蛋白表达系统之一。用于毕赤酵母表达系 统的表达载体种类繁多，却因需要甲醇诱导表达或者基因转化过程中引入抗生素基因而限制了其在食品行业中的 应用。研究设计以毕赤酵母组成型启动子-GAP 启动子替换毕赤酵母表达载体pPIC9 上的甲醇诱导型启动子AOX ， 成功构建了毕赤酵母组成型表达载体pGAPHα。并以里氏木霉木聚糖酶基因xyn2 为报告基因进行功能验证，结果 表明，该载体可实现xyn2 基因在毕赤酵母中的高效表达。在此基础上以经密码子优化后的克鲁维酵母菊粉酶基因 信号肽INU 替换载体上原有的α-Factor 信号肽，结果表明，INU 信号肽能够有效引导XYNII 的分泌。 关键词：毕赤酵母；表达载体；GAP 启动子；INU 信号肽 中图分类号： 文献标识码：A 文章编号：1005－9369 （2009）07－0055－05 Construction and probation of neotype secreting expression vectors for Pichia pastoris /SONG Qingfeng, BAO Lijuan, LI Jie （College of Life Sciences, Northeast Agricul- tural Unive rsity, Harbin 150030, China ) Abstract: P ichia p a stor is is one of the foreign protein expression system which has been widely used. The expression vectors for P ichia p a storis are varied, but they can not be used in food vocation, because of the need of methanol in expression and the leading-in of antibiotic gene by transformation. This research designed to replace the methanol induced AOX promoter by P ichia p a storis s constitutional promoter, GAP promoter, and successfully constructed the secreting expression vector pGAPHα for P ichia p a storis . The gene encoding endo-β -1, 4-xylanase from Trichoderma Re e s e i, and xyn2 gene were used as a reporter gene, and then the function probation indicated that the xyn2 gene could be overexpressed in P ichia p a storis . Based on this neotype