猪源大肠杆菌脂多糖的提取、纯化及活性分析

doi:10.16431/j.cnki.1671-7236.2017.03.040

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (3): 912-919.doi: 10.16431/j.cnki.1671-7236.2017.03.040

猪源大肠杆菌脂多糖的提取、纯化及活性分析

冯将^1,2, 王玉坤^1,2, 魏玉好^1,2, 易琼², 王鲁², 鲜思美¹

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州大学生化工程中心, 贵阳 550025

收稿日期:2016-09-09 出版日期:2017-03-20 发布日期:2017-03-21
通讯作者: 鲜思美 E-mail:xiansimei2005@163.com
作者简介:冯将(1991-),男,贵州石阡人,硕士生,研究方向:预防兽医学,E-mail:fengjianggz@126.com
基金资助:
国家自然科学基金项目（31470128）；贵州省科技厅、省农科院联合基金项目[黔科合LH字（2014）7701]；贵州省科学技术基金项目[黔科合LH字（2014）7667]；贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队[黔科合人才团队（2015）4016]

Extraction, Purification and Activity Analysis of Lipopolysaccharides from Escherichia coli Isolated from Swine

FENG Jiang^1,2, WANG Yu-kun^1,2, WEI Yu-hao^1,2, YI Qiong², WANG Lu², XIAN Si-mei¹

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Biochemistry Engineering Center of Guizhou Province, Guiyang 550025, China

Received:2016-09-09 Online:2017-03-20 Published:2017-03-21

摘要/Abstract

摘要：

为了从猪源大肠杆菌中获得纯度高、产率高的脂多糖（lipopolysaccharides，LPS），并评价其毒力，本试验采集仔猪腹泻粪便作为病料，进行细菌分离鉴定和16S rDNA鉴定，采用热酚水法提取细菌的LPS，通过DNaseⅠ、RNaseA、蛋白酶K和醇沉法对LPS进行纯化，通过苯酚硫酸法、Bradford法和紫外分光光度法测定LPS多糖、蛋白及核酸含量，鲎试剂定量法测定其活性，采用小鼠急性毒性试验方法测定小鼠半数致死量（LD₅₀）。结果显示，分离获得一株具有高致病性的猪源大肠杆菌，纯化的猪源大肠杆菌LPS平均产率为3.74%，其中多糖含量为13.40%，蛋白含量为0.48%，核酸含量为0.06%，主要条带集中在14~160 ku范围内，鲎试剂定量法测定其活性为1.21×10⁷ EU/mg，提取LPS对小鼠的LD₅₀为31.86 mg/kg。该猪场仔猪腹泻是由致病性大肠杆菌引起，获得其毒力因子LPS纯度高、产率高、毒力强，这将为临床防制猪大肠杆菌病及LPS提取纯化提供理论依据。

关键词: 大肠杆菌; 提取纯化; 脂多糖; 急性毒性试验

Abstract:

In order to obtain higher purity and high yield of LPS isolated from Escherichia coli, and evaluate its virulence,excrement of diarrhea piglet was collected as pathological samples, of which, some strains of Escherichia coli were isolated and identified. And furthermore, a strain of Escherichia coli was acquired by 16S rDNA identification. The LPS from this strain of Escherichia coli was isolated with the hot-phenol water method and purified by DNaseⅠ, RNaseA, proteinase K treatment and alcohol precipitation. The polysaccharide, protein and nucleic acid content of purified LPS were detected by phenol-sulfuric acid method, Bradford method and UV points spectrophotometric method. And its bioactivity and acute median lethal dose was determinated by tachypleus amebocyte lysate (TAL) method and acute toxicity experiment method, respectively. The results showed that a strain of highly pathogenic Escherichia coli was successfully isolated. The average yield of purified LPS was 3.74%, the proportion of the polysaccharide was 13.40%, the protein was 0.48%, and the nucleic acid was 0.06%. SDS-PAGE electrophoresis and silver stain showed that the bands were mainly concentrated in the range of 14 to 160 ku. The LAL bioactivity of the prepared LPS was 1.21×10⁷ EU/mg, LD₅₀ of the mouse abdominal cavity was 31.86 mg/kg. The results revealed that the diarrhea piglets in the farm was caused by pathogenic Escherichia coli,purified LPS from this strain of Escherichia coli had the advantages of higher purity, high yield and strong virulence, which would provide theoretical data for clinical prevention and treatment of diarrhea piglets.

Key words: Escherichia coli; isolation and purification; lipopolysaccharide; acute toxicity test

中图分类号:

S852.61

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.

FENG Jiang, WANG Yu-kun, WEI Yu-hao, YI Qiong, WANG Lu, XIAN Si-mei. Extraction, Purification and Activity Analysis of Lipopolysaccharides from Escherichia coli Isolated from Swine[J]. , 2017, 44(3): 912-919.

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猪源大肠杆菌脂多糖的提取、纯化及活性分析

Extraction, Purification and Activity Analysis of Lipopolysaccharides from Escherichia coli Isolated from Swine

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