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目的筛选二价日本血吸虫核酸疫苗质粒的适宜宿主菌,并对其发酵条件进行优化。方法通过综合考察细胞干重、质粒DNA产量及工程菌传代的质粒稳定性等参数,确定适宜宿主菌及其相适应的发酵培养基碳源、氮源和磷酸盐类型;进一步通过中心组合设计响应面试验,优化发酵培养基,并对优化的发酵培养基进行实验验证。结果确定适宜宿主菌为E.coliDH5α,优化的发酵培养基(m/v)为:NaCl1.0%,Yeast Extract1.0%,磷酸盐0.3%[其中m(K2HPO4)∶m(KH2PO4)=1∶4],甘油1.68%,牛肉汁2.28%,豆饼汁4.21%。在此发酵条件下,菌体干重由原来的1.57mg/ml增长至5.68mg/ml,质粒DNA产量由原来的3.94μg/ml增长至36.52μg/ml。结论已筛选出日本血吸虫核酸疫苗质粒适宜宿主菌,并优化了其发酵条件,明显提高了质粒DNA产量,具有大规模发酵生产应用价值。
Objective To screen suitable host strains of bivalent Schistosoma japonicum nucleic acid vaccine plasmid and optimize its fermentation conditions. Methods The carbon sources, nitrogen sources and phosphate types of suitable host bacteria and their corresponding fermentation media were determined by comprehensively examining the parameters such as cell dry weight, plasmid DNA yield and the plasmid stability of the passage of engineering bacteria. Further, Surface test, optimize the fermentation medium, and optimize the fermentation medium for experimental verification. As a result, it was confirmed that the suitable host strain was E. coli DH5α. The optimized fermentation medium (m / v) was: 1.0% NaCl, 1.0% Yeast Extract and 0.3% phosphate [where m (K 2 HPO 4): m : 4], 1.68% of glycerol, 2.28% of beef juice, 4.21% of beancake juice. Under this fermentation condition, the dry weight of mycelia increased from 1.57mg / ml to 5.68mg / ml and the yield of plasmid DNA increased from 3.94μg / ml to 36.52μg / ml. Conclusion The suitable host strain of Schistosoma japonicum nucleic acid vaccine plasmid has been screened, and its fermentation conditions have been optimized, and the yield of plasmid DNA has been remarkably increased. It has the value of large-scale fermentation production.