Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation and Purification of Lipopolysaccharide Derived from Escherichia coli O157:H7 for the Specific Antibody Production

병원성 Escherichia coli O157:H7의 특이 항체 생산을 위한 Lipopolysaccharide분리 및 정제

  • 최학선 (조선대학교 단백질소재센터) ;
  • 신영민 (부산지방식품의약품안전청 시험분석) ;
  • 정숙현 (동서대학교 식품생명공학) ;
  • 박영민 (부산대학교 의과대학 미생물학교) ;
  • 안원근 (경북대학교 생물학과)
  • Published : 2004.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Escherichia coli O157:H7 cause hemorrhagic colitis and the extraintestinal complication of hemolytic-uremic syndrome, with their higher incidence occurring in children. Lipopolysaccharide (LPS) of E. coli O157:H7 is very important to make IgG anti-LPS with bactericidal activity. To identify the characteristic of E. coli OI57:H7, we isolated 60 MDa plasmid and amplified stx genes of shiga-like toxin (Stx) 1, 2 of E. coli O157:H7 by polymerase chain reaction (PCR) method. Using the simple purification method which contained phenol extract, ethanol precipitation and gel filtration steps, the LPS of E. coli O157:H7 was isolated and purified. Finally, we confirmed the purity of LPS through SDS-PAGE and silver nitrate staining.

대장균 O157:H7의 백신 생산을 위해서 purity가 높은 lipopolysacharride 분리, 정제를 위한 실험을 실시하였다. 대장균 O157:H7 균주를 확인하기 위해서 shiga toxin을 생산할 수 있는 60 MDa plasmid를 분리하였고, PCR법에 의해 E. coli O157:H7 shiga-like toxin(Stx) 1, 2의 stx gene을 증폭하여 E. coli O157:H7의 특징 (130 bp, 346 bp)을 확인하였다. E. coli O157:H7 LPS의 분리 정제는 페놀추출, 에탄올 분획 및 gel filtration의 간단한 방법을 사용하였다. 마지막으로 SDS-PAGE와 silver nitrate 염색을 이용하여 LPS의 purity를 확인하였다.

Keywords

References

  1. Johnson WM, Liar H, Bezanson GS. 1983. Cytotoxic Escherichia coli O157:H7 associated with hemorrhagic colitis in Canada. Lancet 1: 76-80.
  2. Morrison DM. 1985. Colony biopsy in verotoxin-induced hemorrhagic colitis and thrombotic thrombocytopenic purpura (TTP). Am J Clin Pathol 86: 108-112.
  3. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, Hebert RJ, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen ML. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 308: 681-685. https://doi.org/10.1056/NEJM198303243081203
  4. Chart H, Rowe B, van der Kar N, Monneas LA. 1991. Serological identification of Escherichia coli O157 as a cause of haemolitic uremic syndrome in Netherlands. Lancet 337: 437- 440.
  5. 日本國立豫防衛生硏究所. 1996. 腸管出血性 大腸菌 O157:H7 の集團發生. 病原微生物檢出情報 17: 180-190.
  6. Kim BU, Okuda J, Matsumoto C, Morigaki T, Asai N, Watanabe H, Nishibuchi M. 1998. Isolation of an Escherichia coli O157:H7 strain producing shiga toxin 1 but shiga toxin 2 from a patient with hemolytic uremic syndrome in Korea. FEMS Microb Lett 166: 43-48. https://doi.org/10.1111/j.1574-6968.1998.tb13181.x
  7. O'Brien AD, Lively TA, Chen ME, Rothman SW, Formal SB. 1983. Escherichia coli O157:H7 stains associated with hemorrhagic coltilis in the United States produce a Shigella dysenteriae 1 (Shiga)-like cytotoxin. Lancet 1: 702-706.
  8. Cohen D, Grcon MS, Block C, Rounch C, Ofek L. 1988. Serum antibodies to lipopolysaccharide and natural immunity to shigellosis in an Israeli military population. J Infect Dis 157: 1068-1071. https://doi.org/10.1093/infdis/157.5.1068
  9. Johnson KG, Perry MB. 1976. Improved techniques for the preparation of bacterial liopolysaccharides. Can J Microbiol 22: 29-34. https://doi.org/10.1139/m76-004
  10. Birnboim HC, Doly J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513-1523. https://doi.org/10.1093/nar/7.6.1513
  11. Wang G, Clark CG, Rodgers FG. 2002. Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 shiga toxin family by multiplex PCR. J Clin Microbiol 40: 3613-3613. https://doi.org/10.1128/JCM.40.10.3613-3619.2002
  12. Smith RJ, BeMiller JN. 1964. Starch. In Methods in Carbohydrate Chemistry. Academic press Inc, New York. Vol IV, p 91-98.
  13. Tsai CM, Frasch CE. 1982. A sensitive silver stain for detection liopopolysaccarides in polyacrylamide gels. Anal Biochem 119: 115-119. https://doi.org/10.1016/0003-2697(82)90673-X
  14. Ostroff SM, Neill MA, Lewis JH, Hargrett-Bean N, Kobayashi JM. 1989. Toxin genotype and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157: H7 infections. J Infect Dis 160: 994-998. https://doi.org/10.1093/infdis/160.6.994
  15. Tzipori S, Karch H, Wachsmuth KI, Robins-Browne RM, O'Brien AD, Lior H, Cohen ML, Smithers J, Levine MM. 1987. Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets. Infect Immun 55: 3117-3125.
  16. Vuddhakul V, Patararungrong N, Pungrasamee P, Jitsurong S, Morigaki T, Asai N, Nishibuchi M. 2000. Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand. FEMS Microbio Letters 182: 343-347. https://doi.org/10.1111/j.1574-6968.2000.tb08919.x
  17. Weinstein DL, Jackson MP, Samuel JE, Holmes RK, O'Brien ADJ. 1998. Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine. J Bacteriol 170: 4223-4230.
  18. Pradel N, Leroy-Setrin S, Joly B, Livrelli V. 2002. Genomic subtraction to identify and characterize sequences of Shigalike toxin producing Escherichia coli O91:H21. Appl Environ Microbiol 68: 2316-2325. https://doi.org/10.1128/AEM.68.5.2316-2325.2002
  19. Lingwood CA, Law H, Richardson S, Petric M, Brunton JL, DeGrandis S, Karmali M. 1987. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J Biol Chem 262: 8834-8839.
  20. Keda M, Gunji Y, Yamasaki S, Takeda Y. 2000. Shiga toxin acivates p38 MAP kinase through cellular $Ca^{2+}$ increase in vero cells. FEBS Letters 485: 94-98. https://doi.org/10.1016/S0014-5793(00)02204-3
  21. Carrion M, Bhat UR, Reuhs B, Carlson RW. 1990. Isolation and characterization of the lipopolysacccharides from Bradyrixobium japonicum. J Bacteriol 172: 1725-1731.
  22. Carlson RW, Lee RP. 1983. A comparison of the surface polysaccharides from Rhizobium leguminosarum 128C53 $sm^rrif^r$ with surface polysaccharudes from its $exo^{-1}mutant^1$. Plant Physiol 71: 223-228. https://doi.org/10.1104/pp.71.2.223
  23. Carlson RW. 1984. Heterogenecity of Rhizobium lipopolysacccharides. J Bacteriol 158: 1012-1017.
  24. Carlson R, Sanders RE, Napoli C, Albersheim P. 1978. Hostsymbiont interactions: purification and partial characterization of Rhizobium lipopolysacccharides. Plant Physiol 62: 912-917. https://doi.org/10.1104/pp.62.6.912
  25. Song SC, Lin L. 1998. Characterization from Rhizobium fredii: Cytostructure, lipopolysacccharides, and cell proteins analysis from Rhizobium fredii. Bot Bull Acad Sin 39: 261-267.
  26. Song SC, Lin LP. 1999. The transition of Rhizobium fredii lipopolysacccharides induced by soybean root exudation. Bot Bull Acad Sin 40: 73-78.