日本血吸虫核酸疫苗质粒适宜宿主菌的筛选及其发酵条件的优化  

Screening of Host Bacterial Strain and Optimization of Fermentation Condition for Production of DNA Vaccine against Schistosoma japonicum

作　　者：朱路[1] 刘海峰[1] 龙全科[1] 鲁明波[1] 余龙江[1] 

机构地区：[1]华中科技大学生命科学与技术学院分子生物物理教育部重点实验室,武汉430074

出　　处：《中国生物制品学杂志》2010年第8期845-851,共7页Chinese Journal of Biologicals

基　　金：教育部博士点基金(20070487059);教育部新世纪优秀人才计划(NCET-06-0646);863项目(2004AA2Z3212)

摘　　要：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 the host bacterial strain of bivalent DNA vaccine against Schistosoma japonicum and optimize the condition for fermentation of the strain. Methods The host bacterial strain as well as the carbon source, nitrogen source and phosphate of fermentation medium were optimized by synthetic evaluation of dry cell weight, plasmid DNA yield and stability of plasmids in subcultured recombinant strain. Fermentation medium was further optimized by central combination design response surface test, then verified. Results E. coli DH5α was screened as the host bacterial strain of DNA vaccine against Schistosoma japonicum. The optimal fermentation medium consisted of（m / v）1. 0% sodium chloride, 1. 0% yeast Extract, 0. 3% phosphate in which the molar ratio of dipotassium hydrogen phosphate to potsssium dihydrogen phosphate was 1 ∶ 4, 1. 68% glycerol, 2. 28% beef juice and 4. 21% bean juice. Under the optimized fermentation condition, the dry cell weight increased from 1. 57 mg / ml to 5. 68 mg / ml, while the plasmid DNA yield from 3. 94 μg / ml to 36. 52 μg / ml. Conclusion The host bacterial strain of DNA vaccine against Schistosoma japonicum was screened, and the fermentation condition was optimized, by which the plasmid DNA yield increased significantly, indicating that the screened host bacterial strain and the optimized fermentation condition were suitable for large scale production of DNA vaccine against Schistosoma japonicum.

关 键 词：血吸虫 日本 疫苗 DNA 质粒 发酵 

分 类 号：R383.24[医药卫生—医学寄生虫学] B92-33[医药卫生—基础医学]