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

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)
Publication Information
Korean Journal of Microbiology / v.44, no.3, 2008 , pp. 178-185 More about this Journal
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.
Keywords
antiserum; avian influenza virus; GST; H9N2; HA; NA; protein expression;
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1 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
2 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   DOI   ScienceOn
3 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   DOI   ScienceOn
4 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   DOI   ScienceOn
5 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   DOI   ScienceOn
6 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
7 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
8 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   PUBMED
9 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   DOI   ScienceOn
10 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   DOI   ScienceOn
11 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   DOI   ScienceOn
12 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
13 Horimoto, T. and Y. Kawaoka. 2005. Influenza: Lessons from past pandemics, warnings from current incidents. Nat. Rev. Microbiol. 3, 591-600   DOI   ScienceOn
14 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   DOI   ScienceOn
15 Alexander, D.J. 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3-13   DOI   ScienceOn
16 Wilson, J.C. and M. Von Itzstein. 2003. Recent strategies in the search for new anti-influenza therapies. Curr. Drug Targets. 4, 389-408   DOI   ScienceOn
17 Harper, S. and D.W. Speicher. 2008. Expression and purification of GST fusion proteins. Curr. Protoc. Protein Sci. Chapter 6: Unit 6.6
18 Alexander, D.J. 2003. Report on avian influenza in the Eastern hemisphere during 1997-2002. Avian Dis. 47, 792-797   DOI   ScienceOn
19 Dolin, R. 2005. Influenza-interpandemic as well as pandemic disease. N. Engl. J. Med. 353, 2535-2537   DOI   ScienceOn
20 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   PUBMED
21 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   DOI   ScienceOn
22 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   DOI
23 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   DOI   ScienceOn
24 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
25 Suzuki, Y. 2005. Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biol. Pharm. Bull. 28, 399-408   DOI   ScienceOn
26 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   DOI
27 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   DOI   ScienceOn
28 Uyeki, T.M. 2008. Global epidemiology of human infections with highly pathogenic avian influenza A (H5N1) viruses. Respirology 13(Suppl 1), S2-S9   DOI   ScienceOn
29 Rosales, J.L. and K.Y. Lee. 2000. Purification of dual-tagged intact recombinant proteins. Biochem. Biophys. Res. Commun. 273, 1058-1062   DOI   ScienceOn
30 Shortridge, K.F. 1992. Pandemic influenza: a zoonosis? Semin. Respir. Infect. 7, 11-25   PUBMED
31 Centers for Disease Control and Prevention. 2005. Key facts about influenza and the influenza vaccine. Centers for Disease Control and Prevention, Atlanta, GA, USA