19F型肺炎链球菌菌种的复壮及其荚膜多糖纯化工艺的优化

目的开发复壮肺炎链球菌19F型(Sreptococcus pneumoniae serotype 19F,简称PN19F)菌种的方法,并优化其荚膜多糖的纯化工艺。方法配制PN19F菌种用液体扩增培养基和固体选择培养基,筛选出荚膜较厚的单菌落,比较复壮前后PN19F菌种的发酵培养情况、纯化后多糖收获量及多糖质量;将发酵液裂解后分别酸沉淀8、24和48 h,并经100 KD超滤纯化,再分别用乙醇法和冻干法制备精制多糖,比较其多糖收获量、组分检定结果、抗原活性、核磁共振图谱。结果两种培养基复壮前后的PN19F菌种,发酵收获菌液A600值及培养时间变化不大。纯化后多糖收获量分别为0. 2和0. 6 g/L,即复壮后多糖收率提高了195%。酸沉淀8、24 h后纯化的多糖质量合格,且24 h较8 h多糖收率高约38%,酸沉淀时间达48 h时,产率无明显变化,但多糖质量不合格。乙醇法和冻干法制备的精制多糖各项指标均合格,且无明显差异。结论应用制备的两种培养基复壮的PN19F菌种,可明显提高荚膜多糖产量。PN19F菌种的酸沉淀时间不超过24 h,乙醇法和冻干法均可用于精制多糖制备。

Rejuvenation of Streptococcus pneumonia serotype 19F and optimization of purification process of capsular polysaccharide

Objective To develop a method for rejuvenating Sreptococcus pneumoniae serotype 19F(PN19F) and optimize the purification process of capsular polysaccharide. Methods Liquid amplification medium and solid selective medium for PN19F were prepared for screening single colonies with thick capsule. The fermentation of PN19F before and after rejuvenating as well as the yield and quality of purified polysaccharides were compared. The fermentation liquid was lysed and subjected to acid precipitation for 8,24 and 48 h,then purified by 100 KD ultrafiltration to obtain polysaccharides by ethanol method and freeze-drying method respectively. The polysaccharide harvest,component test result,antigen activity and ~1H-NMR were compared. Results The A600 value and culture time of fermentation harvest of PN19F before and after rejuvenating showed no significant change. After purification,the yields of purified polysa-ccharides were 0. 2 and 0. 6 g/L respectively,which increased by 195% as compared with those before rejuvenating. The polysaccharides after purification by acid precipitation for 8 and 24 h were qualified,of which the yield after purification for 24 h was about 38% higher than that for 8 h. However,after acid precipitation for 48 h,the yield showed no significant change,while the polysaccharide quality was unqualified. All the indexes of purified polysaccharides prepared by ethanol method and lyophilization method were qualified,which showed no significant difference. Conclusion PN19F strain rejuvenated by two media increased the yield of polysaccharide singificnatly. The acid precipitation time was less than 24 h. Both ethanol method and lyophilization method may be used for the preparation of purified polysaccharide.