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

Abstract

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

CLC Number:

S852.61

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.

References

[1] 马增军, 芮萍, 逯春香,等. 猪源肠外致病性大肠杆菌的分离与鉴定[J]. 中国人兽共患病学报, 2015, 31(2):130-134.
[2] Piotrowska-Tomala K K, Bah M M, Jankowska K, et al. Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland during experimentally induced mastitis in vivo and in vitro[J]. Domestic Animal Endocrinology, 2015, 52(3):90-99.
[3] Kabanov D S, Prokhorenko I R. Structural analysis of lipopolysaccharides from gram-negative bacteria[J]. Biochemistry (Mosc), 2010, 75(4):383-404.
[4] Wang X, Quinn P J. Endotoxins:Structure, function and recognition[J]. Biochemistry, 2011, 76(5):4-5.
[5] 杨成兰, 罗薇, 刘内生,等. 丙型副伤寒沙门氏菌脂多糖提取纯化及活性分析[J]. 中国畜牧兽医, 2014, 41(4):75-78.
[6] Rezania S, Amiromzaffari N, Tabarraei B, et al. Extraction, purification and characterization of lipopolysaccharide from Escherichia coli and Salmonella typhi[J]. Avicenna Journal of Medical Biotechnology, 2011, 3(1):3-9.
[7] Wang X, Heazlewood S P, Krause D O, et al. Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis[J]. Journal of Applied Microbiology, 2003, 95(3):508-520.
[8] 李鹏. 藏猪源大肠杆菌和肠球菌耐药性调查及其耐药机制的研究[D]. 北京:中国农业大学, 2015.
[9] Li Y, Powell D A, Shaffer S A, et al. LPS remodeling is an evolved survival strategy for bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(22):8716-8721.
[10] Wang J, Ma W, Wang Z, et al. Construction and characterization of an Escherichia coli mutant producing Kdo2-lipid A[J]. Marine Drugs, 2014, 12(3):1495-1511.
[11] Wang Z,Wang J,Ren G,et al. Influence of core oligosaccharide of lipopolysaccharide to outer membrane behavior of Escherichia coli[J]. Marine Drugs, 2015, 13(6):3325-3339.
[12] Zhang C, Li Y, Tao G, et al. Characterization of lipid A Cronobacter sakazakii[J]. European Journal of Mass Spectrometry, 2010, 16(4):531-538.
[13] Wang X, Quinn P J. Endotoxins:Lipopolysaccharides of gram-negative bacteria[J]. Sub-cellular Biochemistry, 2011, 76(5):3-25.
[14] Raetz C R H, Whitfield C. Lipopolysaccharide endotoxins[J]. Biochemistry, 2002, 71(71):635-700.
[15] 翟如英. Pseudomonas fragi A22抗逆性的研究及其脂多糖的结构解析[D]. 南京:南京师范大学, 2014.
[16] Wang X, Quinn P J. Purification and characterization of lipopolysaccharides[J].Biochemistry,2011, 76(5):27-50.
[17] 绳秀珍, 孟鹏, 徐晓丽,等. 从许氏平鲉体表分离海分枝杆菌的鉴定及其脂多糖的抗原性分析[J]. 水产学报, 2011, 35(10):1524-1531.
[18] 康洁. 3种方法提取大肠杆菌E.coli脂多糖的比较[J]. 中国农学通报, 2010, 26(21):12-15.
[19] 夏小成, 刘晓颖. 布鲁氏杆菌LPS抗原制备及其免疫原性测定[J]. 特产研究, 2014,1:26-29.
[20] 宋宏新, 刘晓阳, 李宏. 改良热酚水法制备大肠杆菌O157:H7脂多糖抗原的研究[J]. 食品科学, 2006, 27(10):273-275.
[21] 刘红亮, 陈学忠, 李克生,等. 肠出血性大肠杆菌O157:H7脂多糖抗原的提取鉴定及间接ELISA法的建立[J]. 中国人兽共患病学报, 2011, 27(7):637-640.
[22] 郭萌, 李冠民, 黄清泉. 细菌内毒素研究进展[J]. 中国实验动物学报, 2009, 17(5):397-400.
[23] 徐先栋, 谢珍玉, 王世锋,等. 脂多糖快速银染检测方法的改良[J]. 生物技术通报, 2009,25(3):95-97.

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

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