新型Kdo2-lipid A合成菌株构建及其免疫生化活性研究

Construction of Escherichia coli Mutant Producing Novel Kdo2-Lipid A and Study of Its Immunological and Biochemical Activities

摘要:

脂多糖（LPS）是革兰氏阴性细菌外膜的主要组成部分，由疏水的Kdo2-lipid A和亲水的多糖长链组成，能激活先天性免疫系统。Kdo2-lipid A是LPS的生物活性中心，能被开发为新型疫苗佐剂。本研究在前期菌株基础上敲除与LPS庚糖合成相关waaC-waaF基因，构建了直接合成特殊结构Kdo2-lipid A、不含多糖长链的突变株BW003。通过薄层层析和电喷雾电离质谱技术鉴定了BW003合成的Kdo2-lipid A结构；HEK-Blue hTLR4细胞和THP-1细胞刺激实验证实突变株及其合成的Kdo2-lipid A的细胞毒性均明显减弱；生化表型分析结果表明BW003较野生型呈现出更高的外膜渗透性、抗生素敏感性、疏水性及自凝集能力。本研究为后续新型疫苗佐剂开发和产业化利用奠定了理论基础。

Abstract:

Lipopolysaccharides（LPS） is the major component of the outer membrane of Gram-negative bacteria. It's composed of a hydrophobic Kdo2-lipid A and a hydrophilic polysaccharide chain，and can activate the innate immune system. Kdo2-lipid A is the bioactive centers of LPS，which has the potential to be developed as vaccines or vaccine adjuvants. In this study，we constructed a novel Kdo2-lipid A producing mutant BW003 by deleting waaC-waaF genes in the chromosome of a laboratory Escherichia coli strain. The Kdo2-lipid A structure of BW003 was confirmed by TLC and ESI/MS. Compared with wild type E. coli W3110 and LPS，BW003 and Kdo2-lipid A showed lower cytotoxicity when stimulating HEK-Blue hTLR4 cells and THP-1 cells. In addition，BW003 presented higher outer membrane permeability，sensitivity to antibiotics，hydrophobicity and auto-aggregation ability，which is safe and efficient for large-scale industrial fermentation and vaccine adjuvants development.