Long Juan, Zhao Xiao, Liang Fei, Liu Nan, Sun Yuying, Xi Yongzhi
Department of Immunology and National Center for Biomedicine Analysis, Beijing 307 Hospital, No.8, Dongda Ave, Fengtai District, Beijing, 100071 People's Republic of China.
J Biol Eng. 2018 Oct 10;12:22. doi: 10.1186/s13036-018-0110-y. eCollection 2018.
Fermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered DH5α, using the response surface method (RSM).
We hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2 × YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD = 0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.
The present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.
发酵条件优化和营养成分筛选对于高效生产质粒DNA疫苗同等重要。这直接影响质粒DNA疫苗的下游纯化以及最终质量和产量。本研究旨在采用响应面法（RSM）优化工程菌DH5α高通量生产治疗性DNA疫苗pcDNA-CCOL2A1的发酵条件。
我们推测优化的发酵条件可显著提高pcDNA-CCOL2A1治疗性DNA疫苗的产量，该疫苗是一种用于治疗类风湿性关节炎（RA）的新型DNA疫苗。分别进行单因素分析以评估LB、2×YT、TB、M9（甘油）和M9（葡萄糖）这几种基础培养基的最佳配方。此后，采用Plackett-Burman设计（PBD）确定影响疫苗产量的三个最显著因素，随后通过最速上升路径移至最接近最大响应的区域。通过PBD初步筛选发现，最关键的因素是蛋白胨、甘露醇和接种浓度。随后使用响应面法通过Box-Behnken设计（BBD）进一步优化治疗性DNA疫苗pcDNA-CCOL2A1的生产。最终优化的发酵条件如下：蛋白胨25.86 g/L；甘露醇8.08 g/L；接种浓度OD = 0.36。使用这种统计实验设计，在最佳条件下，治疗性DNA疫苗pcDNA-CCOL2A1的产量从223.37 mg/L显著提高到339.32 mg/L，与原始培养基相比提高了51.9%。
本研究结果为中试规模乃至工业规模进一步生产高质量、高产量的治疗性DNA疫苗pcDNA-CCOL2A1提供了依据。
