生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (2): 10-.doi: 10.3969/j.issn.2095-1736.2020.02.010

• 研究报告 • 上一篇    下一篇

包含ARS/CEN元件的游离质粒在巴斯德毕赤酵母中的稳定性研究

  

  1. 江南大学 生物工程学院 粮食发酵工艺与技术国家工程实验室工业生物技术教育部重点实验室， 无锡 214122
  • 出版日期:2020-04-18 发布日期:2020-04-17
  • 通讯作者: 杨艳坤，副教授，硕士生导师，主要从事微生物学、分子生物学和发酵工程研究，E-mail：yangyankun@jiangnan.edu.cn
  • 作者简介:刘国强，硕士，主要从事微生物基因组编辑，E-mail：liuguoqiangzzu@163.com
  • 基金资助:
    国家自然科学基金面上项目(31570034)；江苏省研究生科研创新计划项目(KYCX18_1803, KYCX18_1904)；江苏省第十五批“六大人才高峰”(SWYY-180)

Stability of episomal plasmids containing ARS/CEN  elements in Pichia pastoris

  1. National Engineering Laboratory of Grain Fermentation Technology, Key Laboratory of Industrial Biotechnology, Ministry of Education, College of Bioengineering, Jiangnan University, Wuxi 214122, China
  • Online:2020-04-18 Published:2020-04-17

PDF (PC)

152

摘要： 巴斯德毕赤酵母(Pichia pastoris)是应用最为广泛的外源蛋白表达系统之一。目前使用最多的外源蛋白表达策略是使用整合型质粒；游离型质粒由于其高不稳定性，在毕赤酵母中的应用受到极大的限制。将ARS/CEN等元件连接到pPIC9K载体上，构建能够自主复制的游离型质粒pPIC9K-ARS/CEN，通过传代培养检测质粒丢失的方法探究毕赤酵母在细胞传代培养过程中游离质粒的稳定性。无选择压力情况下，包含ARS/CEN元件的游离质粒在细胞培养10代之前具有极高的稳定性，随后游离质粒开始丢失，到第20代质粒丢失率高达92.94%。该研究结果为构建稳定的游离质粒毕赤酵母表达系统提供了良好的支撑，并提供了切实可行的质粒消除方法，为毕赤酵母运用CRISPR/Cas9系统进行多基因编辑提供了良好的研究基础。

关键词: 巴斯德毕赤酵母, 外源蛋白表达, ARS/CEN, 游离型质粒, 质粒稳定性

Abstract: Pichia pastoris (P. pastoris) is one of the most widely used heterologous protein expression systems. The most widely used heterologous protein expression strategy is the integrated plasmids; the episomal plasmids are greatly limited in Pichia pastoris due to their high instability. In this research, ARS/CEN and other components were ligated into pPIC9K vector to construct autonomously replicating episomal plasmid pPIC9K-ARS/CEN. The way of subculturing to detect plasmid loss was used to investigate the stability of episomal plasmid in Pichia pastoris during cell subculture. In the absence of selective pressure, the episomal plasmid containing the ARS/CEN element had extremely high stability before cell culture for ten generations, and then the episomal plasmid began to be lost, and the loss rate to the 20th generation plasmid was as high as 92.94%. The results of this study provided not only a good support for the construction of a stable episomal plasmid Pichia pastoris expression system, but also a practical plasmid elimination method.

Key words: Pichia pastoris, exogenous protein expression, ARS/CEN, episomal plasmid, plasmid stability

中图分类号: