大肠杆菌生产携带多拷贝重复序列的 DNA 疫苗的发酵工艺

Fermentation Process ofEscherichia coli Producing DNA Vaccine with Multiple Copies of Repetitive Sequences

摘要

携带多拷贝 CpG (Cytosine-phosphate-Guanine) 序列的 DNA 疫苗在水生动物疾病防控领域展现出巨大潜力，但其规模化生产工艺仍是制约产业化的关键瓶颈。本研究以重组大肠杆菌 Stbl3 为工程菌，对携带多拷贝重复序列的质粒 DNA 疫苗发酵工艺进行了优化。通过摇瓶实验筛选确定葡萄糖为最适的碳源种类，最佳质量浓度为 5 g/L；在 5 L 发酵罐中对接种时机、接种量、溶氧水平、补料策略及温度诱导条件等进行了优化，最终确认工艺条件为：于对数中期按 4.5% 接种量接种、维持溶氧水平在 30%、并在发酵后期采用 42 ℃温度诱导。该工艺在 30 L 发酵罐中完成了验证，最终菌体浓度 OD₆₀₀ 达到 42.3，质粒产量提升至 178.6 mg/L。质粒纯化产物经质量检验：OD₂₆₀/OD₂₈₀ 比值处于 1.8~2.0，符合纯度要求；宿主基因组 DNA 与 RNA 污染物含量（质量分数）均低于 1%；质粒超螺旋占比（质量分数）不低于 80%，目标多拷贝重复序列片段经 PCR 验证完整性良好。动物实验表明，该 DNA 疫苗表现出良好的生物安全性；免疫攻毒实验保护率达到 70% 以上。利用重组大肠杆菌 Stbl3 对多拷贝重复序列质粒的发酵工艺优化为水产 DNA 疫苗的产业化发展提供了应用基础。

Abstract

DNA vaccines carrying multiple copies of repetitive sequences show great potential in aquatic animal disease prevention and control, but their large-scale production process remains a key bottleneck hindering industrialization. To achieve high-yield, high-quality plasmid DNA, this study optimized the fermentation process for the recombinantE. coli Stbl3 harboring plasmid DNA vaccines with multiple copies of repetitive sequences. Shaker flask experiments identified glucose as the optimal carbon source at a concentration of 5 g/L. At a 5 L fermenter scale, parameters including inoculation timing, inoculum volume, dissolved oxygen level, feeding strategy, and temperature induction conditions were optimized. The final process conditions were established as follows: inoculation at 4.5% in the mid-log phase, maintaining dissolved oxygen at 30%, and setting induction temperature at 42 ℃ during the late fermentation stage. This process was successfully validated in a 30 L fermenter, yielding a final cell density with an OD₆₀₀ of 42.3 and a maximum plasmid yield reaching 178.6 mg/L. Quality inspection of the purified plasmid products showed that the OD₂₆₀/OD₂₈₀ ratio ranged from 1.8 to 2.0, meeting the purity requirements. The contents of host genomic DNA and RNA contaminants were both below 1%, and the proportion of supercoiled plasmid was no less than 80%. The integrity of the target multi-copy repetitive sequence fragment was verified to be satisfactory by PCR. Animal experiments demonstrated that the DNA vaccine exhibited favorable biosafety, and the protective efficacy in the immune challenge test exceeded 70%. The optimization of the fermentation process for the multi-copy repetitive sequence plasmid using recombinantE. coli Stbl3 lays an application foundation for the industrial development of aquatic DNA vaccines.

关键词

大肠杆菌 Stbl3 / 多拷贝重复序列 / 发酵优化 / 质粒稳定性 / 虹鳟免疫

Key words

E. coli Stbl3 / multiple copies of repetitive sequences / fermentation optimization / plasmid stability / rainbow trout immunity

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基金资助

国家重点研发计划(2023YFC2812102)