无细胞百日咳疫苗生产用培养基的改进和培养条件优化

Improvement of Culture Medium for Production of Acellular Pertussis Vaccine and Optimization of Its Culture Conditions

目的:改进无细胞百日咳疫苗生产用培养基并优化培养条件。方法:用不同浓度溶血(0. 1%)赫氏琼脂及酸水解酪蛋白制备百日咳活性炭培养基,选择最优培养条件;使用百日咳活性炭培养基成功复苏菌种后,优化传代代次和培养时间,评价百日咳活性炭培养基替代羊血包姜氏培养基的可行性;用酸水解酪蛋白代替自制50%酸水解酪蛋白制备改良SS液体培养基,并用ELISA、SDS-PAGE电泳和CHO细胞簇集等方法评价对菌种的培养效果。结果:溶血(0. 1%)赫氏琼脂的加入量为33. 2g/L、酸水解酪蛋白加入量为8g/L时,菌苔生长最快;百日咳活性炭培养基培养第1代72 h、第2代48 h、第3代48 h、第4代24 h和第1代72 h、第2代36 h、第3代24 h的血凝(HA)和UV600均较高,这两组细菌生长状态较好;酸水解酪蛋白制备的百日咳活性炭培养基在优化条件后复苏菌种生长良好,和羊血包姜氏培养基没有明显差异; 8g/L酸水解酪蛋白制备的改良SS液体培养基所得菌量优于50%酸水解酪蛋白制备培养基(P=0. 001),改良SS液体培养基培养所得百日咳毒素纯度、产量和CHO细胞簇集活性均较好。结论:使用酸水解酪蛋白的百日咳活性炭培养基和改良SS液体培养基所得的百日咳杆菌纯度、产量、活性均较高,适用于无细胞百日咳组分疫苗的生产。

Objective： To improve the culture medium for acellular pertussis vaccine production and optimize its culture conditions. Methods： Hottinger＇s Agar of different concentrations and acid hydrolyzed casein were used to prepare pertussis activated carbon medium with optimal culture conditions. After resuscitating the strains with pertussis activated carbon medium, passage and culture time wure optimized and the feasibility of replacing pertussis activated carbon medium with sheep blood-stained medium was evaluated. Acid hydrolyzed casein was used to replace 50% acid hydrolyzed casein in modified SS liquid medium, and the culture effects of the strain were evaluated by ELISA, SDS-PAGE electrophuresis and CHO cell clustering. Results： When Hottingerg Agar was added in an amount of 33.2 g/L and acid hydrolyzed casein was added in an amount of 8 g/L, the lawn glow with the fastest speed. HA and UV600 of pertussis activated carbon medium were higher and bacteria glow better in two sub-cultures-the first generation 72 h, the second generation 48 h, the third generation 48 h, and the fourth generation 24 h and the first generation 72 h, the second generation 36 h, and the third gen- eration 24 h. The pertussis activated carbon medium prepured by acid hydrolysis of casein was well-developed after optimized conditions, and there was no significant difference between the blood and the culture medium of sheep blood. The number of bacteria in the modified SS liquid medium prepared by 8g/L acid hydrolysis casein was better than that of self-made 50% acid hydrolysis casein preparation medium （P = 0. 001 ） , and the purity, yield and bioactivity to CliO cells were good. Conclusion ： The pertussis dried bacteria obtained by the pertussis activated carbon medium using acid hydrolyzed casein and the modified SS liquid medium have higher purity, yield and activity, and were suitable for the production of the acellular pertussis vaccine.