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目的优化重组HIV-1DNA疫苗工程菌的中试发酵工艺。方法优化质粒转化条件,制备工程菌Jm109/pVAX1-MEGp24种子液,连续传30代,检测其遗传稳定性;优化培养基成分、补料基质、补料方式和培养时间,确定最佳发酵参数;并应用最佳发酵参数,于50L发酵罐进行3批中试规模的发酵,考察在发酵培养过程中质粒的稳定性和超螺旋质粒的比例。结果工程菌在连续传代30次后,质粒拷贝数基本保持稳定;最佳发酵参数为:采用以甘油为碳源的培养基,以梯度恒速流加方式补料,培养时间13h;通过3批稳定发酵,最终可获得湿菌67.0～68.6g/L,质粒含量可达1.62～1.73mg/g菌,超螺旋质粒的比例达93%以上。结论已建立了稳定的重组HIV-1DNA疫苗工程菌中试发酵工艺,为进一步规模化生产奠定了基础。
Objective To optimize the pilot fermentation process of recombinant HIV-1 DNA vaccine engineering bacteria. Methods The plasmid transformation conditions were optimized and the engineered bacteria Jm109 / pVAX1-MEGp24 seed solution was prepared. The genetic stability of Jm109 / pVAX1-MEGp24 was determined by continuous passage for 30 passages. The optimum fermentation parameters were determined by optimizing the composition of the medium, feeding matrix, feeding method and culture time. Three batches of pilot scale fermentations were performed in a 50 L fermentor using the optimal fermentation parameters, and the stability of the plasmids and the ratio of supercoiled plasmids during fermentation were investigated. Results The number of plasmid copies remained stable after 30 consecutive passages. The optimum fermentation parameters were as follows: fed with glycerol as carbon source and fed with a constant gradient flow for 13 h; Stable fermentation, finally get wet bacteria 67.0 ~ 68.6g / L, the plasmid content of up to 1.62 ~ 1.73mg / g bacteria, the proportion of supercoiled plasmid up to 93%. Conclusion The pilot fermentation technology of recombinant recombinant HIV-1 DNA vaccine has been established, which laid the foundation for further scale production.