低内毒素大肠杆菌表达体系的构建及应用

孙悦; 陈金平; 苏畅; 李恒; 龚劲松; 许正宏; 史劲松

doi:10.12211/2096-8280.2026-044

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低内毒素大肠杆菌表达体系的构建及应用

研究论文 | 更新时间：2026-07-23

- 低内毒素大肠杆菌表达体系的构建及应用
- Construction and application of low-endotoxin Escherichia coli expression system
- 合成生物学 2026年页码：1-11
- 作者机构：
  
  1.江南大学生命科学与健康工程学院，江苏无锡 214122
  2.江苏集萃未来食品技术研究所有限公司，江苏宜兴 214200
  3.江南大学生物工程学院，粮食发酵与食品生物制造国家工程研究中心，江苏无锡 214122
  4.四川大学轻工科学与工程学院，四川成都 610065
- 作者简介：
  
  孙悦（2000—），女，硕士研究生。研究方向为微生物与生化药学。
  龚劲松（1986—），男，教授，博士，博士生导师。研究方向为医药中间体及功能性化学品绿色生物制造。
- 基金信息：
  
  国家重点研发计划(2024YFA0917900);国家自然科学基金(32571699);江南大学糖化学与生物技术教育部重点实验室开放课题基金(KLCCB-KF202510)
- DOI：10.12211/2096-8280.2026-044
  中图分类号： Q789
- 网络首发：2026-07-23，
- 稿件说明：
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孙悦，陈金平，苏畅，李恒，龚劲松，许正宏，史劲松. 低内毒素大肠杆菌表达体系的构建及应用［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-044

SUN Yue， CHEN Jinping， SU Chang， LI Heng， GONG Jinsong， XU Zhenghong， SHI Jinsong. Construction and application of low-endotoxin Escherichia coli expression system［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-044
孙悦，陈金平，苏畅，李恒，龚劲松，许正宏，史劲松. 低内毒素大肠杆菌表达体系的构建及应用［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-044 DOI：

SUN Yue， CHEN Jinping， SU Chang， LI Heng， GONG Jinsong， XU Zhenghong， SHI Jinsong. Construction and application of low-endotoxin Escherichia coli expression system［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-044 DOI：

摘要

大肠杆菌是常用的重组蛋白表达宿主，但其外膜的内毒素释放可能诱导炎症反应、发热甚至内毒素休克等内毒素相关不良反应，从而限制其在疫苗抗原等医药重组蛋白生产中的应用。本研究旨在构建低内毒素大肠杆菌表达系统，并评估其在猪传染性胸膜肺炎候选抗原重组表达中的应用潜力。以

Escherichia coli

BL21（DE3）为初始菌株，采用CRISPR/Cas9基因编辑技术对核心寡糖、O抗原组装、类脂A修饰及表面多糖合成相关通路进行改造，构建一系列低内毒素候选菌株。相关基因缺失导致初始菌株出现脂多糖结构组装缺陷、胞外多糖中庚糖含量显著下降、脂多糖条带消失和细胞表面粗糙程度增加等现象，其中低内毒素候选菌株ETF13和ETF15的裂解上清液中的内毒素含量分别下降35%、73%。结合内毒素水平和菌株生长状态，ETF13在低内毒素特征与底盘适用性之间表现出较好平衡，因此被选为后续重组蛋白表达宿主。以4个猪传染性胸膜肺炎亚单位疫苗ApxI、ApxII、ApxIII和OMP为模型蛋白，相比于初始菌株，低内毒素候选菌株ETF13的重组蛋白表达能力相当，纯化后的四个蛋白中内毒素残留含量均下降超过50%。综上所述，以上研究结果证明了这种独特的低内毒素大肠杆菌表达体系为医用蛋白的大规模制备提供了新的底盘选择。

Abstract

Escherichia coli

is a commonly used host for recombinant protein expression. However

endotoxins released from its outer membrane may induce endotoxin-related adverse reactions

such as inflammatory responses

fever

and even endotoxin shock

thereby limiting its application in the production of biopharmaceutical recombinant proteins

including vaccine antigens. This study aimed to construct a low-endotoxin

E. coli

expression system and evaluate its application potential in the recombinant expression of candidate antigens against porcine pleuropneumonia. Using

E. coli

BL21(DE3) as the parental strain

CRISPR/Cas9-mediated genome editing was employed to modify pathways related to core oligosaccharide biosynthesis

O-antigen assembly

lipid A modification

and surface polysaccharide synthesis

resulting in a series of low-endotoxin candidate strains. These pathway modifications caused defects in lipopolysaccharide structural assembly

a significant decrease in heptose content in extracellular polysaccharides

disappearance of lipopolysaccharide bands

and increased cell surface roughness. Among the candidate strains

the endotoxin levels in the lysate supernatants of ETF13 and ETF15 decreased by 35% and 73%

respectively. Considering both endotoxin levels and strain growth status

ETF13 showed a favorable balance between low-endotoxin characteristics and chassis applicability and was therefore selected as the host for subsequent recombinant protein expression. Using four subunit vaccine candidate antigens against of

Actinobacillus

pleuropneumoniae

namely ApxI

ApxII

ApxIII

and OMP

as model proteins

ETF13 exhibited recombinant protein expression levels comparable to those of the parental strain. In addition

the residual endotoxin levels in the four purified proteins were reduced by more than 50%. Taken together

these results demonstrate that this engineered low-endotoxin

E. coli

expression system provides a promising alternative chassis for the large-scale production of medical recombinant proteins.

关键词

Keywords

references

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