Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Production of Monoclonal Antibody for Listeria spp. p60 Protein Based on iap Gene

Listeria spp. p60 단백질에 대한 단일클론항체의 생산

  • 임희영 (경기대학교 자연과학부 생물학과 유전학실험실) ;
  • 오연경 (경기대학교 자연과학부 생물학과 유전학실험실) ;
  • 김종수 (경기대학교 자연과학부 생물학과 유전학실험실) ;
  • 이영순 (서울대학교 수의과대학 공중보건학실험실) ;
  • 임윤규 (제주대학교 수의과대학 공중보건학실험실) ;
  • 윤병수 (경기대학교 자연과학부 생물학과 유전학실험실)
  • Published : 2003.03.01
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Abstract

The p60 protein of Listeria spp. is a Listeria-Genus-specific, major extra-cellular protein, which is used as an indicator protein for the detection of these bacteria from contaminated foods. In this study, p60 protein were recombinantly produced in E. coli and were purified using amylose resin based column chromatography. Purified recombinant-p6O was used to generate monoclonal antibody against native p60. Antibody from hybridoma cell line, 1H4, specificically reacted with native p60 protein isolated from pathogenic Listeria spp. such as L. monocytogenes, L. ivanovii, L. welshimeri II, but did not or relatively weakly reacted with non-pathogenic Listeia species, L. innocua or other bacterial proteins. Antibody from 1H4 was produced using ascites fluid method and it may be useful to develop the Listeria-detection kits based on immunological method.

리스테리아의 p60단백질은 Listeria속 고유의 단백질로써, 주요 세포외 단백질이기에, 식품에서 이 세균의 존재를 밝혀주는 중요한 지시단백질로 사용된다. 본 연구는 재조합 DNA 방법으로 재조합-p60 단백질을 대장균에서 생산하였으며, 아밀로오스 컬럼 크로마토그라피의 방법으로 정제하여, Listeria spp.의 p60에 특이적 항체를 생산하는 단일클론을 선별하였다. 생산된 항p60 항체 1H4는 병원성 Listeria 균주인 L. monocytogenes, L. ivanovii, L. weishi meri II 특이적으로 강한 항체반응을 보였으며, Listeria속의 비병원성균인 L. innocua등과는 상대적으로 매우 약한 항체반응을 보였으며, 타 세균의 단백질과는 거의 반응하지 않았다. 1H4항체는 복수생산법에 의해 대량생산되었으며, 이 항체의 특이성은 면역학적 방법에 의한 리스테리아 검출키트의 개발에 유용하게 사용될 수 있을 것이다.

Keywords

References

  1. Ju-Myoung Suk, Ho-Jo Kang, and Won-Geun Son: Direct Detection of Listeria monocytogenes in Chickens by Polymerase Chain Reaction. Kor. J. Vet. Publ. Hlth., 22, 207-214 (1998)
  2. Yoon, B. S., Bubert, A. and Goebel, W.: Rapid detection and application of the Listeria species by polymerase chain reaction (PCR) tools. Federation meeting of Korean basic medical scientists. 3, 409-412 (1995)
  3. Bubert, A., Kohler, S. and Goebel, W: The Homologous and Heterologous Regions within the iap Gene Allow Genus and Species-Specific Identification of Listeria spp. by Polymerase Chain Reaction. Appl. Environ. Microbiol., 58, 2625-2632 (1992)
  4. Bubert, A., Hein, I., Rauch, M., Lehner, A, Yoon, B. S., Goebel, W., and Wagner, M.: Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl. Environ. Microbiol., 65, 4688-4692 (1999)
  5. Cocolin, L., Manzano, M., Cantoni, C. and Cimi, G.: A PCR-microplate capture hybridization method to detect Listeria monocytogenes in blood. Molecular and Cellular Probes, 11, 453-455 (1997) https://doi.org/10.1006/mcpr.1997.0133
  6. Choi, Y. H., Han, S. H., Lee, Y. S., Lim, Y. K, Lim, I. K., and Yoon, B. S.: Expression and purification of Listeria innocua Sv6b p60 invasion associated protein. Biotechnology letters. 23, 1669-1673 (2001) https://doi.org/10.1023/A:1012487425920
  7. Finlay, B. B.: Cracking Listeria's password. Science. 292, 1666-1667 (2001)
  8. Hering, T. M., Kollar, J., Huynh, T. D. and Varelas, J. B.: Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion. Analyt. Biochem., 240, 98-108 (1996) https://doi.org/10.1006/abio.1996.0335
  9. Maina, C. V., Riggs, P. D., Grandea, A. G., Slatko, B. E., Moran, L. S., Tagliamonte, J. A, McReynolds, L. A. and di Guan, C.: An Escherichia coli vector to express and purify foreign proteins by fusion to, and separation from maltose binding protein. Gene, 74, 365-373 (1988) https://doi.org/10.1016/0378-1119(88)90170-9
  10. Park, J. H., Choi, E. A., Cho, E. W, Hahm, K. S. and Kim, K. L.: Maltoes binding protein (MBP) fusion proteins with low or no affinity to amylose resins can be single-step purified using a novel anti-MBP monoclonal antibody. Mol. cells, 8, 709-716 (1998)
  11. Rijpens, N. P., Jannes, G., Asbroeck, M. V., Herman, L. M. F. and Rossau, R.: Simultaneous detection of Listeria spp. and Listeria monocytogenes by reverse hybridization with 16S-23S rRNA spacer probes. Molecular and Cellular Probes, 9, 423-432 (1995) https://doi.org/10.1006/mcpr.1995.0065
  12. Park, J. H., Lee, Y. S., Lim, Y. K., Kwon, S. H., Lee, C. U. and Yoon, B. S.: Specific binding of recombinant Listeria monocytogenes p60 protein to Caco-2 cells. FEMS Microbiology Letters 186, 35-40 (2000) https://doi.org/10.1111/j.1574-6968.2000.tb09078.x
  13. Lim, H. Y., Lee, Y. S., Lim, Y. K., Kang, K. S. and Yoon, B. S.: Molecular cloning and Expression of MBP-p60 proteins of pathogenic Listeria species. Biotechnology letters (Submitted) (2002)