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

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.