肺炎球菌菌种复苏用无动物源性固体培养基的筛选

Screening of solid culture medium without animal-derived materials for resuscitation of Streptococcus pneumoniae

目的 筛选一种可复苏肺炎球菌(Streptococcus pneumoniae,Spn)菌种的无动物源性物料的固体培养基。方法 首先选取19F血清型Spn对无动物源材料的9种配方培养基进行初步筛选,再对其他23个疫苗血清型Spn进行培养测试验证。通过对传代15代的培养物进行菌种检定与荚膜多糖抗原基因序列分析,判断筛选的配方培养基对各型菌种传代稳定性的影响。用此培养基复苏的菌种按生产程序进行发酵培养,分析培养情况及荚膜多糖抗原产量及质量,确定筛选培养基的生产适用性。结果 在筛选的配方固体培养基上,各型Spn培养11~15h时活菌数量较高,15代终末代次的菌种检定、抗原基因序列与对照(羊血固体培养基培养的菌种)相比无差异。用其复苏的菌种发酵培养后,菌体生长与荚膜多糖产量略有提高,且荚膜多糖相关检定指标均符合《中国药典》三部(2020版)标准。结论 筛选的无动物源性固体培养基具有良好的适用性,可取代血液来源培养基用于Spn疫苗生产菌种复苏。

Objective To screen a kind of solid culture medium without animal-derived materials for resuscitating Streptococcus pneumoniae(Spn).Methods Spn 19F was selected for the preliminary screening of 9 kinds of formula culture media without animal-derived materials,and then other 23 serotypes of Spn were cultured for verification.The effect of the selected medium on the hereditary stability of various serotypes was identified by strain identification and capsular polysaccharide antigen gene sequence analysis of the 15-generation strains.The strains resuscitated by this medium were fermented according to the production procedure,and the culture situation and the yield and quality of capsular polysaccharide antigen were analyzed to determine the production applicability of the screened medium.Results The number of viable bacteria of various types of Spn was desired after 11~15 h of incubation on the selected solid medium and there was no difference in strain identification and antigen gene sequence between the final 15 generation and the control(strains cultured in sheep blood solid medium).After fermentation and culture of the strain resuscitated by this medium,the cell growth and the capsular polysaccharide production increased slightly,and all the related verification indicators of capsular polysaccharide met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Conclusion The selected solid culture medium without animal-derived materials has good applicability and can replace blood-derived culture medium for the resuscitation of Spn strain for vaccine production.