生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 43-.

• 研究报告 • 上一篇    下一篇

谷氨酸棒杆菌中基于5′UTR及其下游序列的增强型表达载体构建

  

  1. 1.江南大学 粮食发酵工艺与技术国家工程实验室， 无锡 214122;
    2.江南大学 工业生物技术教育部重点实验室， 无锡 214122;
    3.江南大学 生物工程学院， 无锡 214122
  • 出版日期:2021-10-18 发布日期:2021-10-21
  • 通讯作者: 白仲虎，教授，研究方向为生物医药蛋白的高效表达及发酵过程研究，E-mail: baizhonghu@jiangnan.edu.cn
  • 作者简介:方求武，硕士研究生，研究方向为生物大分子结构、功能与制备，E-mail: kevinf957@outlook.com;高　雄，硕士研究生，研究方向为分子微生物学，E-mail: 735984473@qq.com；方求武和高　雄为共同第一作者
  • 基金资助:
    国家自然科学基金项目（编号21808082,21878124）

Construction of enhanced expression vector based on 5′UTR and its downstream sequence in Corynebacterium glutamicum

  1. 1. National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi 214112, China;
    2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;
    3. School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2021-10-18 Published:2021-10-21

PDF (PC)

48

摘要： 从谷氨酸棒杆菌CGMCC1.15647基因组中鉴定蛋白丰度最高基因的5′UTR(5′-untranslated region)及其下游序列，并利用这些5′UTR及其下游序列与两种高强度启动子PH36和Ptac组合分别构建一系列单顺反子和双顺反子表达载体。5′UTR及其下游序列显著提升启动子的表达强度，其中表达强度最高的pTac-B2826-EGFP荧光强度是阳性对照pTac-Positive的3.6倍。使用筛选的增强型表达载体在谷氨酸棒杆菌中成功地表达VHH蛋白（variable domain of heavy chain of heavy-chain antibody），在摇瓶发酵水平，VHH的分泌表达量达到85.4 mg/L。筛选的双顺反子载体可成为谷氨酸棒杆菌内源基因过表达和重组蛋白生产的有利工具。

关键词: 谷氨酸棒杆菌, 5′UTR, 双顺反子表达载体, VHH

Abstract: Several endogenous 5′UTRs (5′-untranslated region) and their downstream sequences with highest protein abundance were firstly identified from the genome of Corynebacterium glutamicum CGMCC1.15647, and these 5′UTRs and their downstream sequences with two strong promoters PH36 and Ptacwere used to construct monocistronic (MEM) and bicistronic expression model (BEM). In both MEM and BEM, the 5′UTR and its downstream sequence significantly enhanced the expression ability of these promoters. Among them, the highest expression intensity of these vector, pTac-B2826-EGFP, was about 3.6 times that of the positive expression vector pTac-Positive. Several vectors with high expression ability were selected to express the variable domain of heavy chain of heavy-chain antibody (VHH) protein in Corynebacterium glutamicum. In the shake flask fermentation culture, the secreted expression of the protein VHH reached 85.4 mg/L. All these works provided several new vectors with strong expression ability for heterologous protein expression of Corynebacterium glutamicum.

Key words: Corynebacterium glutamicum, 5′UTR, BEM, VHH

中图分类号: