研制14型肺炎链球菌培养基和优化其荚膜多糖提纯的工艺

Develop a culture medium for Streptococcus pneumonia serotype 14 and improved the purification of its capsular polysaccharide

目的研制国产培养14型肺炎链球菌的培养基,然后制备和纯化该菌的荚膜多糖,并对其全过程的工艺进行优化。方法测试添加不同种血清、培养基成分对肺炎链球菌生长的影响,在此基础上对比进口培养基和本实验自制培养基培养细菌的效果。测试不同裂解细菌方案对获得荚膜多糖多少的影响,并比较DNA酶和RNA酶酶解方法和常规乙醇沉淀方法去除残留核酸的效果。结果实验室自配培养基与进口培养基均能使肺炎链球菌较好生长。用1%NP40加胰酶的裂解方法可使多糖从细菌菌体更好的释放,使用DNA酶和RNA酶去除残留核酸,在多糖产量和核酸去除效率方面都取得了较好的结果,再进一步纯化后可以得到较纯的多糖。结论本文在培养基国产化、菌体裂解后多糖释放和核酸去除三大方面为肺炎球菌荚膜多糖提取提供了有效可行的方案。

Objective To develop a culture medium for Streptococcus pneumonia serotype 14 and purify it capsular polysaccharide （CPS）. Methods Different serums,different medium components were added to estimate the growth condition of the bacteria.Based on these results,the homemade medium was compared with the import medium for culturing this strain.Then,the quantities of CPS obtained from different methods of cracking bacteria were measured. Different from the traditional chemical method,our method was adopted enzyme to remove nucleic acid and phenol to extract proteins for purifying the CPS. Results It was indicated that no matter Streptococcus pneumonia was cultivated in the prepared medium or in the import medium; they could both reach the best growth condition. Besides, it was good for releasing CPS with 1% NP40 to crack bacteria. They can get more CPS and the nucleic acid was removed better by using DNase and RNase. Moreover, it could obtain more pure CPS with further purification. Conclusion Above all,this paper provides a feasible and effective solution to purify the CPS in three ways which were included the optimal condition of medium formula, bacteria cracking and polysaccharide release and nucleic acid removal.