재조합 Shigatoxin 2e 유전자의 발현단백 생산 및 독소의 수용체 결합 친화성 확인

Production of expressed protein from cloned ShigatoxinG 2e gene and Receptor Binding Affinity of the toxin

  • 동분연 (녹십자백신연구소) ;
  • 김상현 (캐나다 구엘프대학교 수의과대학) ;
  • 김영일 (수의과학검역원 부산지소) ;
  • 조현호 (수의과학검역원 서울지소) ;
  • 이우원 (부산시 보건환경 연구원) ;
  • 김곤섭 (경상대학교 수의과대학 생명과학연구소) ;
  • 강호조 (경상대학교 수의과대학 생명과학연구소) ;
  • 김용환 (경상대학교 수의과대학 생명과학연구소)
  • Dong, Bun-youn (Korea Green Cross Co.) ;
  • Kim, Sang-Hyun (Department of Pathobiology, Ontario Veterinary College, University of Guelph) ;
  • Kim, Yeong-Il (National Veterinary Research and Quarantine Service) ;
  • Cho, Hyun-Ho (National Veterinary Research and Quarantine Service) ;
  • Lee, Woo-won (Pusan Institute of Health and Environment) ;
  • Kim, Kon-Sup (College of Veterinary Medicine, Research Institute of Life Science,Kyeongsang National University) ;
  • Kang, Ho-Jo (College of Veterinary Medicine, Research Institute of Life Science,Kyeongsang National University) ;
  • Kim, Yong-Hwan (College of Veterinary Medicine, Research Institute of Life Science,Kyeongsang National University)
  • 심사 : 2004.04.17
  • 발행 : 2004.06.30

초록

This study was designed to determine optimal condition for expression of cloned Shigatoxin2e(Stx2e) gene from transformed E. coli PED18, to compare the cytotoxicity titer between cloned Stx2e and Stx2e from original strain, and to confirm of receptor binding affinity of Stx2e for use of development of receptor binding ELISA to detect of Stx2e. The optimum composition of medium for expression of Stx2e gene in E.coli host-vector system was definded as the medium containing 0.5% glucose and 0.5 mM IPTG. The cytotoxicity titer of expressed Stx2e for Vero cell was 1000 fold higher than that of Stx2e from original strain AY93258. The binding affinity of Stx2e to receptor globotetraosyl ceramide($Gb_4$) was confirmed by immunobloting.

참고문헌

  1. 김용환, 차인호, 조현호, 김상현, 진형근, 김영일, 김도. Verocytotoxin 생성 대장균의 분포 및 Receptor를 이용한 RELISA법의 개발. II. Verotoxin의 검출을 위한 Receptor Binding Enzyme-Iinked Immunesorbent Assay(RELISA)에 관하여. 한국수의공중보건학회지. 1996, 20, 55-63
  2. 김용환, Lingwood, C. A. Verotoxin 생성 대장균의 병원성에 관한 연구. 한국수의공중보건학회지. 1992, 16, 215-221
  3. 김종희, 차인호, 김용환. 부종병감염 돼지 유래 대장균 O139로 부터 Verotoxin-E 유전자의 클로닝한 염기서열 분석. 한국수의공중보건학회지. 1997, 21, 281-288
  4. 차인호, 김용환. 동물분변에서 Escherichia coli O157:H7의 분리 및 이들 균이 생산하는 Verotoxin-2의 생물화학적 특성. II. 동물분변에서 분리한 Escherichia coli O157:H7으로부터 Verotoxin-2의 정제 및 특성. 대한수의사회지. 1996, 36, 379-387
  5. Basta, M., Karmali, M. A. and Lingwood, C. A. Sensitive receptor specified enzyme-linked immunosorbent assay for Escherichia coli verotoxin. J. Clin. Microbiol. 1989, 27, 1617-1622
  6. Boyd, B. and Lingwood, C. A. Verotoxin receptor glycolipid in human renal tissue. Nephron. 1989, 51, 207-210
  7. Cerny, G. and Teuber, M. Differential release of periplasmic versus cytoplasmic proteins from Escherichia coli by polymyxin B. Arch. Microbiol. 1971, 179, 166-179
  8. Frank, M. A., Jorsal S. E., Ahrens, P., Tensen, N. E. and Meyling A. Molecular characterization of Escherichia coli strains isolated from pigs with edemadisease, J. elin, Microbiol. 1997, 35, 20-24
  9. Franke, S., Harmsen, D., Caeprioli, A., Pierard, D., Wieler, L. H. and Carch, H. Clonal relatedness of shiga-like toxin producing Esherichia coli O-101 strainsof human and porcine orgin. J. Clin. Microbiol. 1995, 33, 3174-3178
  10. Frydendahl, K. Prevalence of serogroups and virulence in Escherichia coli associated with postweaning diarrhea and edema disease in pigs and a comparison of diagnostic approaches. Vet. Microbiol. 2002, 85, 169-182
  11. Gentry, M. K. and Dalrymple. J. M. Quantitative microtiter cyototoxicity assay for shiga toxin. J. Clin. Microbiol. 1980, 12, 361-366
  12. Gyles, C. I. DeGrandis, S. A., Mackenzie, C. and Brunton, C. J. Cloning and nucleotide sequence analysis of the genes determinig Verocytotoxinproduction in a porcine edema disease isolates of E. coli. Microbial. Pathogen. 1988, 5, 419-426
  13. Ling, H., Pannus, N. S., Boodhoo, A., Armstrong, G. D., Clark, C. G., Brun, J. L. and Read, R. J. A mutant shiga-like toxin IIe bound to its receptor Gb(3)structure of a group II shiga-like toxin with altered bind specificity. Structure Fold Des. 2000, 8, 253-264
  14. Lingwood, C. A. Role of verotoxin receptors in pathogenesis. Trends in Microbiol. 1996, 4, 147-152
  15. Macleod, D. L. and Gyles, C. L. Purification and characterization of an Escherchia coli shiga-like Toxin II varient. Infect. Immun. 1990, 58, 1232-1239
  16. MacLeod, D. L., Gyles, C. L. and Wilcock, B. P. reproduction of edema disease of swine with purified shiga-like toxin II variant. Vet. Pathol. 1991, 28, 66-73
  17. Mary, G. K. and Joel, M. D. Microtiter cytotoxicity assay for shigella toxin. J. Clin. Microbiol. 1982, 12, 362-367
  18. Methyiapun, S., Pohlenz, J. F. L. and Bertschinger, H. U. Ultrastructure of the intestinal mucosa in pigs experimentally in oculated with an edema diseaseproducing strain of Escherichia coli (O139:K12:H1). Vet. Pathol. 1984, 21, 516-520
  19. Moxley, R. A. Edema disease. Vet. Clin. North Am. Food Anim. Pract. 2000, 16, 175-185
  20. Petric, H. M., Karmali, M. A., Richardson, S. and Cheung, R. Purification and biological prorerties of Escherichia coli verocytotoxin. FEMS. Microbiol. Lett.1987, 41, 63-68
  21. Samuel, J. E., Perera, L. P., Ward, S., O'Brien, A. D., Ginsburg, V. and Krivan, H. C. Comparison of the glycolipid receptor specificities of the Shiga-like toxin type II and Shiga-like toxin type II variants. Infect. Immun. 1990, 58, 611-618
  22. Silva, A. S., Valadares, G. F., Penatti, M. P. A., Brito, B. G. and Leit, D. S. Escherichia coli strains from edema disease : O serogroups and genes for shiga toxin, enterotoxin, and F18 fimbriae. Vet. Microbiol. 2001, 80, 227-233
  23. Weinstein, D. L., Jackson, M. P., Samuel, J. E., Holmes, R. K. and O'Brien, A. D. Cloning and sequencing of a shiga-like toxin type II variant from an Escherichia coli strain responsible for edema disease of swine. J. Bacteriol. 1988, 170, 4223-4230