抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究

Efficient Soluble Expression of Anti-IgE scFv in E.coli and Optimization of Expression Conditions

目的:以抗IgE单链抗体(anti-IgE scFv)为研究对象,以大肠杆菌周质高效表达可溶性单链抗体为目标,研究不同宿主细胞、培养基及培养条件对可溶性单链抗体表达产量的影响。方法:构建Rosetta(DE3)、BL21(DE3)和SoluBL21(DE3)三种大肠杆菌工程菌,研究不同碳源、氮源、培养基配方和培养条件对可溶性抗IgE单链抗体产量的影响。结果:携带抗IgE单链抗体基因的pET-IgE26质粒在新型宿主SoluBL21(DE3)中的表达产量明显优于传统的Rosetta(DE3)和BL21(DE3)宿主菌。经碳氮源筛选、培养基和培养条件的优化,确定SoluBL21(DE3)工程菌高效表达可溶性重组抗体的最佳培养基为:以M9培养基为基础,添加0.5%葡萄糖、0.6%酪蛋白胨和0.02%微量元素。最佳的培养条件为:以LB为种子培养基,37℃过夜培养至OD600为3.0左右,以5%接种量接种于最佳发酵培养基中;接种后细胞生长条件:37℃,260r/min,培养3.5h;细胞诱导培养条件:当OD600为1.5～1.8时,降温至25℃,添加0.1mmol/L IPTG,220r/min继续培养16h。经抗原ELISA检测,抗IgE单链抗体的可溶性表达量比原始对照组提高了5倍。结论:通过对宿主细胞类型的筛选、培养基及培养条件的优化,可以显著提高重组单链抗体在大肠杆菌周质空间内的表达产量。该研究结果不仅为规模化制备抗IgE单链抗体提供了技术支持,同时为大肠杆菌生产重组小分子抗体提供了有价值的参考。

Objective： An anti-IgE scFv was used to investigate the influence of different host strains, growth media, and culture conditions on the high-level expression of soluble single-chain antibody fragment in E. coli periplasm. Method： Three engineered bacterial strains, Rosetta （ DE3 ）, BL21 （ DE3 ）, and SoluBL21 （ DE3 ）, were constructed and effects of different carbon sources, nitrogen sources, growth media, and culture conditions on the amount of expression of soluble anti-IgE seFv were evaluated. Results： The expression level of pET-IgE26, a plasmid that carried anti-IgE scFv sequence, was significantly enhanced in the novel host strain SoluBL21 （ DE3 ）, compared to that in traditional Rosetta （ DE3 ） and BL21 （ DE3 ） host strains. After optimization of carbon and nitrogen sources, growth media and culture conditions, the optimal growth media for high-level expression of soluble recombinant antibodies in SoluBL21 （ DE3 ） engineered strain was found to be M9 medium with 0.5% glucose, 0.6% bacto casitone, and 0.02% trace elements. The best culture condition was defined as growing overnight culture in LB medium at 37℃, until ODoreached approximately 3.0, then inoculate the optimal growth media 260r/min for 3.5h. For induction, with the overnight culture at 5% inoculum concentration, shake at 37℃, lower the temperature to 25 ℃ when OD600 is between 1.5 - 1.8, add 0.1 mmol/L IPTG, and incubate at 220r/min for 16h. ELISA detected that the expression of soluble anti-IgE scFv increased by 5-fold after optimization. Conclusion： Expression of recombinant scFv in E coli periplasm can be significantly enhanced through optimization of host strain types, growth media, and culture conditions. The study provides technical support for scale-up production of anti-IgE scFv and insights into the production of recombinant micromolecular antibody by E. coil