基于重组技术的低毒力高效大肠杆菌原核表达系统的改造与应用

背景]大肠杆菌作为原核表达系统常用宿主菌株,具有培养成本低、周期短和操作性强等优势,但同时也存在着一些不足。目的]降低大肠杆菌内毒素合成水平及毒力,同时提高其可溶性表达外源蛋白的能力。方法]利用CRISPR-Cas技术敲除大肠杆菌BL21(DE3)脂多糖生物合成途径中的lpxM,改变其毒性中心类脂A的侧链结构;在基因组中整合表达tig提供蛋白折叠所需伴侣因子;构建pET-28a-Rcodon重组载体,补充蛋白表达所需稀有密码子对应的tRNAo结果]lpxM缺失后菌体细胞内毒素合成水平较出发菌株降低90%左右;利用改造后的重组菌株和载体,可显著提高鸡传染性法氏囊病病毒VP2蛋白的可溶性表达水平;临床安全性试验结果表明,重组菌株BL21(DE3)ΔlpxM::tig合成内毒素的毒力水平较出发菌株显著降低,表达抗原对应免疫组个体无任何临床免疫副反应出现。结论]利用重组技术对大肠杆菌原核表达系统进行改造,并用于外源蛋白的低毒力高效可溶性表达,为相关亚单位疫苗的研究奠定了一定的基础。

Improving Escherichia coli prokaryotic expression system with low virulence and high efficiency by recombination technology

Background]As a common host used in prokaryotic expression system,Escherichia coli owned many advantages like low cultivation cost,short growth period and strong operability while also shared some deficiencies isochronally.Objective]To decrease the endotoxin biosynthesis and virulence of E.coli and improve its exogenous proteins soluble expression ability synchronously.Methods]lpxM gene in lipopolysaccharide biosynthesis pathway of E.coli was primarily deleted by CRISPR-Cas technology for lipid A side chain structure modification.Then tig gene was integrated expressed in genome of E.coli BL21(DE3)ΔlpxM to provide chaperon factor for exogenous proteins.Besides,the recombinant plasmid pET-28 a-Rcodon was constructed to supply the rare codon relevant tRNA for protein expression.Results]Bacterial somatic endotoxin level obtained a 90%reduction compared to the original strain after lpxM deletion.By utilizing the recombinant host strain and plasmid,expression level of the infectious bursal disease virus(IBDV)VP2 protein was observably enhanced.Clinical safety evaluation results indicated that the endotoxin virulence of E.coli BL21(DE3)△lpxM::tig was apparently lower than the original strain and the relevant immunity group showed no clinical symptoms.Conclusion]E.coli prokaryotic expression system was remolded by recombinant technologies for low virulence and high soluble expression of the exogenous proteins,which laid a certain foundation for the relevant subunit vaccines investigation.