乙肝治疗性可复制型DNA疫苗pSVK-HBVA高稳定宿主菌的筛选及其基础培养基的选择

摘要

Objective To screen a high stable host bacterium that can repeatedly transfer the culture and meet the re -quirements of pilot-scale technology and to select the optimal basic fermentation medium for this engineering bacterium . Methods A plasmid pSVK-HBVA was transformed into several different E. coli competent cells and the morphology of ex -tracted plasmids was monitored by agarose gel electrophoresis , with the continuous passage method used for testing the sta -bility of the engineered bacterium. Based on the above -mentioned results , the engineered bacterium that harbored a plasmid with the highest yield and stability was selected as the original seed to establish the three seed lots and identify the seed lots. From LB, TB, M9 ( glycerol) and M9 ( glucose) we selected the optimal medium which could generate the highest plasmid yield. Results We found that plasmid pSVK-HBVA showed similar stability to conventional plasmid DNA harbored in E. coli XL10-Gold host bacterium, which could transfer 30 passages without recombination or fragment loss and meet the requirements of the pilot process . LB was the optimal basic fermentation medium that could generate the highest plasmid volumetric yield 5. 5 mg/L. Conclusion The host bacterium E. coli XLIO-Gold is more conducive to amplification of self replication HBV therapeutic DNA vaccine pSVK-HBVA, and the optimal basic fermentation medium for this engineered bacterium is LB.%目的 为乙肝治疗性可复制型DNA疫苗质粒pSVK-HBVA筛选稳定性高,能多次传代的宿主菌,以满足中试工艺的要求,同时对该工程菌最适的基础发酵培养基进行选择.方法 将质粒pSVK-HBVA分别转化几种不同的大肠杆菌感受态细胞并提取质粒,通过琼脂糖凝胶电泳检测质粒的形态结构,连续传代法对工程菌的稳定性进行检测;选取质粒含量最高和稳定性最好的工程菌作为原始种子,按照2010版<中国药典>要求建立工程菌的三级种子批,并对种子批进行鉴定.同时,从LB,TB,M9(甘油),M9(葡萄糖)4种培养基中为工程菌筛选质粒产量最高的基础发酵培养基.结果 经过筛选发现,在以大肠杆菌XL10-Gold为宿主菌时,质粒pSVK-HBVA表现出了同常规质粒DNA类似的稳定性,转接传代30次也未发生重组或片段丢失,能满足后续中试工艺的要求,LB作为基础培养基能得到最高的质粒容积产率为5.5 mg/L.结论 大肠杆菌XL10-Gold是一个更利于可复制型DNA疫苗质粒pSVK-HBVA扩增的宿主菌,该工程菌发酵的最佳基础培养基是LB培养基.