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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 culturing Streptococcus pneumoniae type 14 in China and then to prepare and purify the capsular polysaccharide of the strain and to optimize its whole process. Methods The effects of adding different kinds of serum and culture medium on the growth of Streptococcus pneumoniae were tested. On the basis of this, the effect of culture on imported culture medium and culture medium in this experiment was compared. The effects of different solutions of lytic bacteria on the amount of capsular polysaccharide obtained were tested, and the effect of residual DNA removal by DNAase and RNase enzymatic methods and conventional ethanol precipitation methods was compared. Results Laboratory self-formulated medium and imported medium can make Streptococcus pneumoniae better growth. Using 1% NP40 plus trypsin cleavage method can make the polysaccharide release better from bacterial cells, the use of DNase and RNase to remove residual nucleic acid, polysaccharide yield and nucleic acid removal efficiency have achieved good results, and then After purification can be more pure polysaccharide. Conclusion This article provides an effective and viable solution for the extraction of pneumococcal capsular polysaccharide in three aspects: culture medium localization, release of polysaccharides after cell lysis and nucleic acid removal.