The Extraction of Vibrio vulnificus Lipopolysaccharide by the Phenol-Chloroform-Petroleum Ether Method and Biological Activity of the LPS

Kim, Ju Seob; Park, Seok Don

Abstract

BACKGROUND: Vibrio vulnificus is a pathogenic, marine, halophilic, gram-negative bacillus which causes fulminant infecticn in humans through the ingestion of raw seafood or skin wounds. V. vulnificus produces seveal kinds of virulent factors including cytolysin, endotoxin, exoenzymes, and siderphores. Among these, the lipopolysaccharide(LPS) of V. vulnificus has recently been purified, but the biological activity of this endotoxin is not well clarified as yet.
OBJECTIVE
The purpose of this study was to extract LPS from Vibrio vulnificus and to test the biological activity of extracted LPS for the elucidation of the role in V. vulnificas septicemia.
METHODS
V. vulnificus LPS was extracted by the Phenol Chloroform-Petroleurn ether(PCP) method. The biological activity of LPS was evaluated with a limulus amebocyte lysate assay and by assessment of lethality to ICR mice.
RESULTS
Five hundreds mg of LPS were extracted from 10g of dried V. vulnificus. Lirnulus amebocyte lysate formed a hard gel in response to the extracted LPS. This LPS showed low level of protein contaminatior in SDS-PAGE electophoresis and spectrophotometry. A High dose of LPS(200 mg/ml body weight) was lethal to mice.
CONCLUSION
The PCP extraction yield relatively large amounts of LPS from V. vnlnificus. with out significant protein contamination and e xtrated LPS has endotoxin activity. This extrated LPS can be used for further studies such as making antibody or characterizing pathogenic roles in the V. vulnificus infection.