论文部分内容阅读
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。
The purpose of this study was to screen the host strain of hepatitis B therapeutic replicable DNA vaccine plasmid pSVK-HBVA with high stability and multiple passages to meet the requirement of pilot process, and to select the optimum fermentation medium for the engineered bacteria. Methods Plasmid pSVK-HBVA was transformed into several different E.coli competent cells and plasmids were extracted respectively. The morphological structure of plasmids was detected by agarose gel electrophoresis. The stability of engineering bacteria was detected by continuous passage method. The best stability of the engineering bacteria as the original seed, according to the 2010 edition of “Chinese Pharmacopoeia” to establish three-level seed of engineering bacteria batch, and identification of the seed batch. At the same time, the engineered bacteria were screened from the four kinds of medium of LB, TB, M9 (glycerol) and M9 (glucose) for the highest yield of basic fermentation medium. Results After screening, it was found that plasmid pSVK-HBVA showed similar stability to that of conventional plasmid DNA when E. coli XL10-Gold was used as the host strain, and no recombination or fragment loss occurred in the passage 30 passages, The requirements of the test process, LB as the basal medium to get the highest plasmid volume yield was 5.5 mg / L. Conclusion Escherichia coli XL10-Gold is a host strain that is more conducive to the replication of the recombinant DNA vaccine plasmid pSVK-HBVA. The optimal medium for the fermentation of this engineering strain is LB medium.