Fermentation process of Escherichia coli Stbl3 producing DNA vaccine with multiple copies of repetitive sequences

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 recombinant E. coli strain 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. In 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 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, achieving a final cell density of OD₆₀₀ 42.3 and plasmid yield to 178.6 mg/L. Quality inspection of the purified plasmid products showed that the A260/A280 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 reached above 70%. The optimization of the fermentation process for the multi-copy repetitive sequence plasmid using recombinant Escherichia coli Stbl3 lays an application foundation for the industrial development of aquatic DNA vaccines.