Expression, Purification and Antiserum Production of the Avian Influenza H9N2 Virus HA and NA Proteins

Avian Influenza H9N2 Virus의 HA와 NA 단백질 발현, 정제 및 항혈청 생산

  • Lee, Hyun-Ji (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Song, Byung-Hak (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Jeong-Min (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Yun, Sang-Im (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Jin-Kyoung (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kang, Young-Sik (Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Koo, Yong-Bum (School of Biotechnology and Biomedical Science, Inje University) ;
  • Jeon, Ik-Soo (Research Planning Team, National Institute of Animal Science) ;
  • Byun, Sung-June (Animal Biotechnology Division, National Institute of Animal Science) ;
  • Lee, Youn-Jeong (National Veterinary Research and Quarantine Service) ;
  • Kwon, Jun-Hun (National Veterinary Research and Quarantine Service) ;
  • Park, Jong-Hyeon (National Veterinary Research and Quarantine Service) ;
  • Joo, Yi-Seok (National Veterinary Research and Quarantine Service) ;
  • Lee, Young-Min (Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University)
  • 이현지 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 송병학 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 김정민 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 윤상임 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 김진경 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 강영식 (충북대학교 의과대학 미생물학교실.기초의학연구소) ;
  • 구용범 (인제대학교 의생명공학대학 생명공학부) ;
  • 전익수 (농촌진흥청 축산과학원 축산기획조정과) ;
  • 변승준 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 이윤정 (국립수의과학검역원) ;
  • 권준헌 (국립수의과학검역원) ;
  • 박종현 (국립수의과학검역원) ;
  • 주이석 (국립수의과학검역원) ;
  • 이영민 (충북대학교 의과대학 미생물학교실)
  • Published : 2008.09.30

Abstract

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

조류 독감바이러스(avian influenza virus, AIV)는 사람에게서 발생하는 인플루엔자 대유행에 중요한 역할을 한다. 특히 최근 AIV H9N2형에 의한 가금류 감염이 빈번히 나타나고 있어 인체 감염이 상당히 우려되는 실정이다. 본 연구에서는 최종적으로 AIV의 HA와 NA 단백질에 특이적으로 반응하는 항혈청을 생산하고자 하였다. 먼저 감염된 닭에서 분리된 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96의 게놈RNA로부터 RT-PCR 방법으로 HA와 NA 단백질 N-말단부위에 해당하는 염기서열을 증폭하였다. 이렇게 증폭된 DNA단편은 E. coli 발현벡터 pGEX4T-1에 삽입한 후, BL21 세포에서 각각의 GST fusion protein (GST-HAln와 GST-NAn) 형태로 발현하였다. GST-HAln와 GST-NAn은 모두 glutathione sepharose column을 사용하여 분리 및 정제하였으며, 정제된 단배질을 항원으로 사용하여 토끼 항혈청을 생산하였다. 생산된 항혈청의 항원특이성은 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96로 감염된 MDCK 세포의 cell extract를 사용하여 immunoblotting을 수행함으로써 확인하였다. 본 실험결과AIV H9N2의 HA와 NA단백질 N-말단부위에 해당하는 재조합GST fusion protein과, 이들 각각의 단백질에 특이적으로 반응하는 항혈청은 앞으로AIV 감염의 진단 뿐만 아니라, AIV에 대한 기초연구에 중요한 재료로 사용될 것으로 기대한다.

Keywords

References

  1. Alexander, D.J. 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3-13 https://doi.org/10.1016/S0378-1135(00)00160-7
  2. Alexander, D.J. 2003. Report on avian influenza in the Eastern hemisphere during 1997-2002. Avian Dis. 47, 792-797 https://doi.org/10.1637/0005-2086-47.s3.792
  3. Banks, J., E.C. Speidel, P.A. Harris, and D.J. Alexander. 2000. Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype. Avian Pathol. 29, 353-359 https://doi.org/10.1080/03079450050118485
  4. Centers for Disease Control and Prevention. 2005. Key facts about influenza and the influenza vaccine. Centers for Disease Control and Prevention, Atlanta, GA, USA
  5. Dolin, R. 2005. Influenza-interpandemic as well as pandemic disease. N. Engl. J. Med. 353, 2535-2537 https://doi.org/10.1056/NEJMp058276
  6. Fouchier, R.A., V. Munster, A. Wallensten, T.M. Bestebroer, S. Herfst, D. Smith, G.F. Rimmelzwaan, B. Olsen, and A.D. Osterhaus. 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79, 2814-2822 https://doi.org/10.1128/JVI.79.5.2814-2822.2005
  7. Gambotto, A., S.M. Barratt-Boyes, M.D. De Jong, G. Neumann, and Y. Kawaoka. 2008. Human infection with highly pathogenic H5Nl influenza virus. Lancet 371, 1464-1475 https://doi.org/10.1016/S0140-6736(08)60627-3
  8. Ghedin, E., N. Sengamalay, M. Shumway, J. Zaborsky, T. Feldblyum, V. Subbu, D. Spiro, J. Sitz, H. Koo, P. Bolotov, D. Dernovoy, T. Tatusova, Y. Bao, K.S. George, J. Taylor , D. Lipman, C. Fraser, J. Taubenberger, and S. Salzberg. 2005. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature 437, 1162-1166 https://doi.org/10.1038/nature04239
  9. Guo, Y.J., S. Krauss, D.A. Senne, I.P. Mo, K.S. Lo, X.P. Xiong, M. Norwood, K.F. Shortridge, R.G. Webster, and Y. Guan. 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 267, 279-288 https://doi.org/10.1006/viro.1999.0115
  10. Harper, S. and D.W. Speicher. 2008. Expression and purification of GST fusion proteins. Curr. Protoc. Protein Sci. Chapter 6: Unit 6.6
  11. Horimoto, T. and Y. Kawaoka. 2005. Influenza: Lessons from past pandemics, warnings from current incidents. Nat. Rev. Microbiol. 3, 591-600 https://doi.org/10.1038/nrmicro1208
  12. Kim, G.Y., M.K. Nam, S.S. Kim, H.Y. Kim, S.K. Lee, and H.A. Rhim. 2008. Simple and rapid strategy for the molecular cloning and monitoring of mouse HtrA2 serine protease. Biotechnol. Lett. 30, 397-403 https://doi.org/10.1007/s10529-007-9556-6
  13. Kung, N.Y., R.S. Morris, N.R. Perkins, L.D. Sims, T.M. Ellis, L. Bissett, M. Chow, K.F. Shortridge, Y. Guan, and M.J. Peiris. 2007. Risk for infection with highly pathogenic influenza A virus (H5N1) in chickens, Hong Kong 2002. Emerg. Infect. Dis. 13, 412-418 https://doi.org/10.3201/eid1303.060365
  14. Kung, N.Y., Y. Guan, N.R. Perkins, L. Bissett, T. Ellis, L. Sims, R.S. Morris, K.F. Shortridge, and J.S. Peiris. 2003. The impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in Hong Kong. Avian Dis. 47, 1037-1041 https://doi.org/10.1637/0005-2086-47.s3.1037
  15. Lin, Y.P., M. Shaw, V. Gregory, K. Cameron, W. Lim, A. Klimov, K. Subbarao, Y. Guan, S. Krauss, K. Shortridge, R. Webster, N. Cox, and A. Hay. 2000. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc. Natl. Acad. Sci. USA 97, 9654-9658
  16. Liu, J., K. Okazaki, H. Ozaki, Y. Sakoda, Q. Wu, F. Chen, and H. Kida. 2003. H9N2 influenza viruses prevalent in poultry in China are phylogenetically distinct from A/quail/Hong Kong/G1/97 presumed to be the donor of the internal protein genes of the H5N1 Hong Kong/97 virus. Avian Pathol. 32, 551-560 https://doi.org/10.1080/0307-9450310001596728
  17. Matrosovich, M.N., S. Krauss, and R.G. Webster. 2001. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281, 156-162 https://doi.org/10.1006/viro.2000.0799
  18. Palese, P. and M.L. Shaw. 2007. Orthomyxoviridae: The viruses and their replication, p. 1647-1689. In D.M. Knipe and P.M. Howley (eds.), Fields Virology, 5th ed., vol. 2. Lippincott Williams & Wilkins Publishers, Philadelphia, USA
  19. Peiris, J.S., Y. Guan, D. Markwell, P. Ghose, R.G. Webster, and K.F. Shortridge. 2001. Cocirculation of avian H9N2 and contemporary 'human' H3N2 influenza A viruses in pigs in southeastern China: potential for genetic reassortment? J. Virol. 75, 9679-9686 https://doi.org/10.1128/JVI.75.20.9679-9686.2001
  20. Peiris, M., K.Y. Yuen, C.W. Leung, K.H. Chan, P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge. 1999. Human infection with influenza H9N2. Lancet 354, 916-917 https://doi.org/10.1016/S0140-6736(99)03311-5
  21. Rosales, J.L. and K.Y. Lee. 2000. Purification of dual-tagged intact recombinant proteins. Biochem. Biophys. Res. Commun. 273, 1058-1062 https://doi.org/10.1006/bbrc.2000.3063
  22. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
  23. Senne, D.A., J.E. Peason, and B. Pahigrahy. 1992. Live poultry markets: a missing link in the epidemiology of avian infl uenza. Proceedings of the Third International Symposium on Avian Influenza; May 27-29
  24. Shortridge, K.F. 1992. Pandemic influenza: a zoonosis? Semin. Respir. Infect. 7, 11-25
  25. Shortridge, K.F., W.K. Butterfield, R.G. Webster, and C.H. Campbell. 1977. Isolation and characterization of infl uenza A viruses from avian species in Hong Kong. Bull. World Health Organ. 55, 15-20
  26. Suzuki, Y. 2005. Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biol. Pharm. Bull. 28, 399-408 https://doi.org/10.1248/bpb.28.399
  27. Ubach, J., Y. Lao, I. Fernandez, D. Arac, T.C. Sudhof, and J. Rizo. 2001. The C2B domain of synaptotagmin I is a $Ca^{2+}$-binding module. Biochemistry 40, 5854-5860 https://doi.org/10.1021/bi010340c
  28. Uyeki, T.M. 2008. Global epidemiology of human infections with highly pathogenic avian influenza A (H5N1) viruses. Respirology 13(Suppl 1), S2-S9 https://doi.org/10.1111/j.1440-1843.2007.01224.x
  29. Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Chambers, and Y. Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152-179
  30. Wilson, J.C. and M. Von Itzstein. 2003. Recent strategies in the search for new anti-influenza therapies. Curr. Drug Targets. 4, 389-408 https://doi.org/10.2174/1389450033491019
  31. Wright, P.F., G. Neumann, and Y. Kawaoka. 2007. Orthomyxoviruses, p. 1692-1740. In D.M. Knipe and P.M. Howley (eds.), Fields Virology, 5th ed., vol. 2. Lippincott Williams & Wilkins Publishers, Philadelphia, USA