屎肠球菌荚膜多糖的提取纯化及其多糖含量的测定

Extraction,Purification and Determination of Capsular Polysaccharide in Enterococcus faecium

为提取纯化屎肠球菌(E.faecium)荚膜多糖(CPS)并测定其多糖含量,试验采用高压法破碎菌体,加入核糖核酸酶(25μg/mL)、脱氧核糖核酸酶(0.5mg/mL)、溶菌酶(0.25mg/mL)及蛋白酶K(1.25mg/mL)等去除核酸和蛋白质,得到荚膜多糖粗提物,经过30ku和100ku超滤离心管超滤浓缩和DEAE Sepharose Fast Flow层析柱层析后,得到纯化的屎肠球菌荚膜多糖,再利用苯酚-硫酸法测定其含量。结果表明,成功从屎肠球菌中提取纯化出荚膜多糖,其含量为75.21%,初步确定了屎肠球菌荚膜多糖的提取纯化方法,为今后相关疫苗的研制奠定了基础。

In order to extract and purify the capsular polysaccharide of Enterococcus faecium and measure contents of polysaccharide,high pressure crushing methods and enzyme decomposition methods（RNase A（25μg/mL）,DNase（0.5mg/mL）,lysozyme（0.25mg/mL）and protease K（1.25mg/mL））were used to remove the nucleic acids and proteins to obtain crude capsular polysaccharide,and centrifugal ultrafiltration with 30 and 100ku ultrafiltration centrifuge tubes and DEAE Sepharose Fast Flow column chromatography were also adopted to obtain the purified capsular polysaccharides of Enterococcus faecium.Then the content of polysaccharide was determined by the phenol-sulfuric acid method.The results showed the capsular polysaccharide content was75.21%.The results revealed that capsular polysaccharide of Enterococcus faecium was extracted successfully.The method of extracting capsular polysaccharide in Enterococcus faecium was established initially.