屎肠球菌荚膜多糖的提取纯化及其多糖含量的测定

doi:10.16431/j.cnki.1671-7236.2017.05.008

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (5): 1309-1314.doi: 10.16431/j.cnki.1671-7236.2017.05.008

屎肠球菌荚膜多糖的提取纯化及其多糖含量的测定

刘永安, 李学瑞, 周建华, 马丽娜, 刘永生

中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点试验室, 农业部草食动物疫病重点开放试验室, 兰州 730046

收稿日期:2016-10-08 出版日期:2017-05-20 发布日期:2017-05-18
通讯作者: 李学瑞, 刘永生 E-mail:lixuerui@caas.cn;liuyongsheng@caas.cn
作者简介:刘永安(1991-),男,山东济宁人,硕士生,研究方向:动物传染病病原学与流行病学,E-mail:457659155@qq.com
基金资助:
国家自然科学基金（31172335）

Extraction,Purification and Determination of Capsular Polysaccharide in Enterococcus faecium

LIU Yong-an, LI Xue-rui, ZHOU Jian-hua, MA Li-na, LIU Yong-sheng

Key Laboratory of Grazing Animal Diseases of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2016-10-08 Online:2017-05-20 Published:2017-05-18

摘要/Abstract

摘要：

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（25 μg/mL）、脱氧核糖核酸酶（0.5 mg/mL）、溶菌酶（0.25 mg/mL）及蛋白酶K （1.25 mg/mL）等去除核酸和蛋白质，得到荚膜多糖粗提物，经过30 ku和100 ku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。

关键词: 屎肠球菌; 荚膜多糖; 提取纯化; 苯酚-硫酸法

Abstract:

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 (25 μg/mL), DNase (0.5 mg/mL), lysozyme (0.25 mg/mL) and protease K (1.25 mg/mL)) were used to remove the nucleic acids and proteins to obtain crude capsular polysaccharide, and centrifugal ultrafiltration with 30 and 100 ku 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 was 75.21%. The results revealed that capsular polysaccharide ofEnterococcus faecium was extracted successfully. The method of extracting capsular polysaccharide in Enterococcus faecium was established initially.

Key words: Enterococcus faecium; capsular polysaccharide; extraction and purification; phenol-sulfuric acid method

中图分类号:

S852.61

刘永安, 李学瑞, 周建华, 马丽娜, 刘永生. 屎肠球菌荚膜多糖的提取纯化及其多糖含量的测定[J]. 《中国畜牧兽医》, 2017, 44(5): 1309-1314.

LIU Yong-an, LI Xue-rui, ZHOU Jian-hua, MA Li-na, LIU Yong-sheng. Extraction,Purification and Determination of Capsular Polysaccharide in Enterococcus faecium[J]. , 2017, 44(5): 1309-1314.

参考文献

[1] 张哲,李新圃,杨峰,等.荚膜多糖及其疫苗研究进展[J].动物医学进展,2015,36(11):83-87.
[2] 杨学云,吴建勇,李建军,等.金黄色葡萄球菌5型荚膜多糖偶联抗原的制备及免疫原性分析[J].中国兽医科学,2012,42(3):299-303.
[3] 周琪,贺现辉,阳巧梅,等.副猪嗜血杆菌SC096株荚膜多糖抗猪肺泡巨噬细胞吞噬能力的研究[J].畜牧兽医学报,2016,47(6):1232-1238.
[4] 吕存女,陈作江,赵海平,等.一种新的CTAB加入方法对A群脑膜炎球菌荚膜多糖分子大小的影响[J].微生物学免疫学进展,2014,42(5):38-42.
[5] 周琪,贺现辉,杜雅萍,等.副猪嗜血杆菌SC096株荚膜多糖及其抗血清的制备[J].中国畜牧兽医,2014,41(12):81-84.
[6] 陈作江,冯鹏,赵海平,等.A群脑膜炎球菌荚膜多糖纯化工艺的优化[J].微生物学免疫学进展,2014,42(5):33-37.
[7] 韦鹏翀,魏茂琪,常亚军,等.Triton X-114萃取法纯化b型流感嗜血杆菌荚膜多糖的应用[J].药物生物技术,2016,23(4):296-298.
[8] Mehta A, Zydney A L. Permeability and selectivity analysis for ultrafiltration membranes[J]. Membrane Science, 2005, 249(1-2):245-249.
[9] Kothari S, Kothari N, Lee E, et al. Development of an efficient and scalable method for processing and purification of Vi capsular polysaccharide[J]. Procedia in Vaccinology, 2010, 2(1):78-81.
[10] Huebner J, Wang Y, Krueger W A,et al. Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Entercoccusfaecium[J].Infection and Immunity, 1999, 67(3):1213-1219.
[11] Ali L, Spiess M, Wobser D,et al.Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317[J].Infection, Genetics and Evolution, 2016, 37:215-224.
[12] 杨育芳,梁琳琳,孙雪南,等.B群链球菌荚膜多糖抗原的分离纯化工艺研究[J].中国生化药物杂志.2006,27(3):166-169.
[13] Weiss E, Long J L. Simplified method for the production of carbohydrateantigen from Neisseria meningitides[J].Applied Microbiology,1969,18(5):843-847.
[14] Tanizaki M M, Garcia L R, Ramos J B, et al. Purification of meningococcal group C polysaccharide by a procedure suitable for scaleup[J]. Microbiological Methods,1996,27(1):19-23.
[15] 姬泽薇,陈守义,毕晓云,等.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[J].热带医学杂志.2012,12(4):394-397.
[16] Pato T P, Barbosa A P R, Silva J G J. Purification of capsular polysaccharidefrom Neisseria meningitidesserogroup C by liquid chromatograph[J]. Chromatography B, 2006, 832(2):262-267.
[17] Kao G, Tsai C M. Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD)[J]. Vaccine,2004,22(3-4):335-344.
[18] Ricci S, Bardotti A, Ascenzi S D, et al. Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection[J]. Vaccine, 2001,19(15-16):1989-1997.
[19] Lei Q P, Lamb D H, Heller R, et al. Quantitation of low level unconjugated polysaccharide in tetanus toxoid-conjugate vaccine by HPAEC/PAD following rapid separation by deoxycholate/HCl[J]. Journal of Pharmaceutical and Biomedical Analysis,2000, 21(6):1087-1091.

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 11

编辑推荐

Metrics

本文评价

[1]	白伟琴, 李宏, 孟克, 訾占飞, 李杰, 赵利军, 王金玲, 格日勒图, 李琦, 吉林台. 1株羊源致病性屎肠球菌的分离与鉴定[J]. 中国畜牧兽医, 2021, 48(10): 3889-3895.
[2]	毛俊舟, 董丽, 王淑楠, 彭众, 仲召鑫, 喻礼怀. 屎肠球菌对AA肉鸡血清和肝脏抗氧化功能的影响[J]. 《中国畜牧兽医》, 2018, 45(8): 2182-2189.
[3]	彭众, 董丽, 王淑楠, 毛俊舟, 韩冬, 喻礼怀. 日粮中添加屎肠球菌对AA肉鸡生产性能、免疫器官指数和血液脂质代谢相关指标的影响[J]. 《中国畜牧兽医》, 2018, 45(6): 1502-1509.
[4]	解伟纯, 王玉峰, 皮若冰, 赵东方, 姜艳平, 崔文, 乔薪瑗, 王丽, 周晗, 徐义刚, 李一经, 唐丽杰. 表达猪乳铁蛋白肽重组屎肠球菌对断奶仔猪生长性能的影响及其抗ETEC感染效果研究[J]. 《中国畜牧兽医》, 2018, 45(12): 3408-3418.
[5]	冯将, 王玉坤, 魏玉好, 易琼, 王鲁, 鲜思美. 猪源大肠杆菌脂多糖的提取、纯化及活性分析[J]. 《中国畜牧兽医》, 2017, 44(3): 912-919.
[6]	刘永安, 李学瑞, 周建华, 马丽娜, 刘永生. 一株羊源致病性屎肠球菌的分离鉴定及耐药性分析[J]. 《中国畜牧兽医》, 2017, 44(2): 530-537.
[7]	张芬芬, 李学瑞, 杨建设. 屎肠球菌新序列型ST989菌株的分离鉴定及多重耐药性分析[J]. , 2015, 42(5): 1274-1282.
[8]	周琪, 贺现辉, 杜雅萍, 樊惠英, 廖明. 副猪嗜血杆菌SC096株荚膜多糖及其抗血清的制备[J]. , 2014, 41(12): 81-84.
[9]	王凡, 王建设, 刘婷, 李晓清, 王安如. 一株猪源屎肠球菌的分离、鉴定及益生功能初步研究[J]. , 2012, 39(7): 231-234.
[10]	华均超, 张邦辉, 周明, 李金友. 屎肠球菌T013复合制剂饲喂生长猪的效果研究[J]. , 2012, 39(10): 112-116.
[11]	赵彦宗;贺东生;苏丹萍. 猪胸膜肺炎放线杆菌血清3型间接血凝试验检测方法的建立[J]. , 2010, 37(11): 143-145.