Production of H5N1 Hemagglutinin inTrichoplusia ni Larvae by a Novel Bi-cistronic Baculovirus Expression Vector
Highly pathogenic avian influenza (HPAI) H5N1 viruses have created demand for a cost-effective vaccine to prevent a pandemic of the disease. Here, we report that Trichoplusia ni (T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an potential effective vaccine for chickens. To facilitate the recombinant virus identification, virus titer determination and access the infected larvae, we employed the internal ribosome entry site (IRES) derived from Perina nuda virus (PnV, belongs to insect picorna like Iflavirus genus) to construct a bi-cistronic baculovirus expression vector that can express the rH5HA protein and enhanced green fluorescent protein (EGFP) simultaneously. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa H5HA1) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for H5N1 vaccine production.
[1] M. F. Ducatez, , C. M Olinger, A. A. Owoade, Z. Tarnagda, M. C. Tahita,
A. Sow, S. De Landtsheer, W. Ammerlaan, J. B. Ouedraogo, A. D.
Osterhaus, R. A. Fouchier, and C. P. Muller, Molecular and antigenic
evolution and geographical spread of H5N1 highly pathogenic avian
influenza viruses in western Africa. J. Gen. Virol. 88 ( 2007) 2297-2306.
[2] M. Gilbert, X. Xiao, J. Domenech, J. Lubroth, V. Martin, and J.
Slingenbergh, Anatidae migration in the western Palearctic and spread of
highly pathogenic avian influenza H5NI virus. Emerg. Infect. Dis. 12
(2006) 1650-1656.
[3] J. J. Treanor, J. D. Campbell, K. M. Zangwill, T. Rowe, M. Wolff, Safety
and immunogenicity of an inactivated subvirion influenza A (H5N1)
vaccine. N. Engl. J. Med. 354 (2006) 1343-1351.
[4] X. Lu, L. E. Edwards, J. A. Desheva, D. C. Nguyen, A. Rekstin, I.
Stephenson, K. Szretter, N. J. Cox, L. G. Rudenko, A. Klimov, J. M. Katz,
Crossprotective immunity in mice induced by live-attenuated or
inactivated vaccines against highly pathogenic influenza A (H5N1)
viruses. Vaccine 24 (2006) 6588-6593.
[5] K. Wang, K. M. Holtz, K. Anderson, R. Chubet, W. Mahmoud, M. M. J.
Cox, Expression and purification of an influenza hemagglutininÔÇöone
step closer to a recombinant protein-based influenza vaccine. Vaccine 24
(2006) 2176-2185.
[6] K. Subbarao, C. Luke, H5N1 viruses and vaccines. PLoS Pathog. 3 (2007)
e40. doi:10.1371/journal.ppat.0030040.
[7] Y. J. Lin, M. C. Deng, S. H. Wu, Y. L. Chen, H. C. Cheng, C. Y. Chang,
M. S. Lee, M. S. Chien, and C.C. Huang, Baculovirus-Derived
Hemagglutinin Vaccine Protects Chickens from Lethal Homologous
Virus H5N1 Challenge. J. Vet. Med. Sci. 70 (2008) 1147-1152.
[8] Y. J. Chen, W. S. Chen, T. Y. Wu, Development of a bi-cistronic
baculovirus expression vector by the Rhopalosiphum padi virus 5' internal
ribosome entry site. Biochem. Biophys. Res. Commun. 335(2005)
616-623.
[9] D. R. O'Reilly, L. K. Miller, V. A. Luckow, Baculovirus Expression
Vector: A Laboratory Manual. W.H. Freeman, NY, 1992.
[10] M. S. Lee, M. C. Deng, Y. J. Lin, C. Y. Chang, H. K. Shieh, J. Z. Shiau,
and C. C. Huang, Characterization of an H5N1 avian influenza virus from
Taiwan. Vet. Microbiol. 124 (2007)193-201.
[11] World Health Organization Global Influenza Program Surveillance
Network. Evolution of H5N1 avian influenza viruses in Asia. Emerg.
Infect. Dis. 11(2005) 1515-1521.
[12] T. R. Jinn, S. S. Kao, Y. C.Tseng, Y. J. Chen, and T. Y. Wu, Aerosol
infectivity of a baculovirus to Trichoplusia ni larvae: an alternative larval
inoculation strategy for recombinant protein production. Biotechnology
Progress. 25 (2009) 384-9.
[13] E. Martinez-Salas, Internal ribosome entry site biology and its use in
expression vectors. Curr. Opin. Biotechnol., 10 (1999) 458-464.
[14] J. Kuzio, D.Z. Rohel, C.J. Curry, A. Krebs, E.B. Carsten, and P. Faulkner,
Nucleotide sequence of the p10 polypeptide gene of Autographa
californica nuclear polyhedrosis virus. Virology, 139 (1984) 414-418.
[15] H.J. Cha, T. Gotoh, and W.E. Bentley, Simplification of titer
determination for recombinant baculovirus by green fluorescent protein
marker. BioTechniques, 23 (1997) 782-786.
[16] C. Ranking, B.F. Ladin, and R.F. Weaver, Physical mapping of
temporally regulated, overlapping transcripts in the region of the 10K
protein gene in Autographa californica nuclear polyhedrosis virus. J.
Virol., 57 (1986) 18-27.
[17] T. Y. Wu, C. Y. Wu, Y. J. Chen, C. Y. Chen, and C. H. Wang, The 5'
untranslated region of Perina nuda virus (PnV) possesses a strong internal
translation activity in baculovirus-infected insect cells. FEBS Lett. 581
(2007) 3120-3126.
[1] M. F. Ducatez, , C. M Olinger, A. A. Owoade, Z. Tarnagda, M. C. Tahita,
A. Sow, S. De Landtsheer, W. Ammerlaan, J. B. Ouedraogo, A. D.
Osterhaus, R. A. Fouchier, and C. P. Muller, Molecular and antigenic
evolution and geographical spread of H5N1 highly pathogenic avian
influenza viruses in western Africa. J. Gen. Virol. 88 ( 2007) 2297-2306.
[2] M. Gilbert, X. Xiao, J. Domenech, J. Lubroth, V. Martin, and J.
Slingenbergh, Anatidae migration in the western Palearctic and spread of
highly pathogenic avian influenza H5NI virus. Emerg. Infect. Dis. 12
(2006) 1650-1656.
[3] J. J. Treanor, J. D. Campbell, K. M. Zangwill, T. Rowe, M. Wolff, Safety
and immunogenicity of an inactivated subvirion influenza A (H5N1)
vaccine. N. Engl. J. Med. 354 (2006) 1343-1351.
[4] X. Lu, L. E. Edwards, J. A. Desheva, D. C. Nguyen, A. Rekstin, I.
Stephenson, K. Szretter, N. J. Cox, L. G. Rudenko, A. Klimov, J. M. Katz,
Crossprotective immunity in mice induced by live-attenuated or
inactivated vaccines against highly pathogenic influenza A (H5N1)
viruses. Vaccine 24 (2006) 6588-6593.
[5] K. Wang, K. M. Holtz, K. Anderson, R. Chubet, W. Mahmoud, M. M. J.
Cox, Expression and purification of an influenza hemagglutininÔÇöone
step closer to a recombinant protein-based influenza vaccine. Vaccine 24
(2006) 2176-2185.
[6] K. Subbarao, C. Luke, H5N1 viruses and vaccines. PLoS Pathog. 3 (2007)
e40. doi:10.1371/journal.ppat.0030040.
[7] Y. J. Lin, M. C. Deng, S. H. Wu, Y. L. Chen, H. C. Cheng, C. Y. Chang,
M. S. Lee, M. S. Chien, and C.C. Huang, Baculovirus-Derived
Hemagglutinin Vaccine Protects Chickens from Lethal Homologous
Virus H5N1 Challenge. J. Vet. Med. Sci. 70 (2008) 1147-1152.
[8] Y. J. Chen, W. S. Chen, T. Y. Wu, Development of a bi-cistronic
baculovirus expression vector by the Rhopalosiphum padi virus 5' internal
ribosome entry site. Biochem. Biophys. Res. Commun. 335(2005)
616-623.
[9] D. R. O'Reilly, L. K. Miller, V. A. Luckow, Baculovirus Expression
Vector: A Laboratory Manual. W.H. Freeman, NY, 1992.
[10] M. S. Lee, M. C. Deng, Y. J. Lin, C. Y. Chang, H. K. Shieh, J. Z. Shiau,
and C. C. Huang, Characterization of an H5N1 avian influenza virus from
Taiwan. Vet. Microbiol. 124 (2007)193-201.
[11] World Health Organization Global Influenza Program Surveillance
Network. Evolution of H5N1 avian influenza viruses in Asia. Emerg.
Infect. Dis. 11(2005) 1515-1521.
[12] T. R. Jinn, S. S. Kao, Y. C.Tseng, Y. J. Chen, and T. Y. Wu, Aerosol
infectivity of a baculovirus to Trichoplusia ni larvae: an alternative larval
inoculation strategy for recombinant protein production. Biotechnology
Progress. 25 (2009) 384-9.
[13] E. Martinez-Salas, Internal ribosome entry site biology and its use in
expression vectors. Curr. Opin. Biotechnol., 10 (1999) 458-464.
[14] J. Kuzio, D.Z. Rohel, C.J. Curry, A. Krebs, E.B. Carsten, and P. Faulkner,
Nucleotide sequence of the p10 polypeptide gene of Autographa
californica nuclear polyhedrosis virus. Virology, 139 (1984) 414-418.
[15] H.J. Cha, T. Gotoh, and W.E. Bentley, Simplification of titer
determination for recombinant baculovirus by green fluorescent protein
marker. BioTechniques, 23 (1997) 782-786.
[16] C. Ranking, B.F. Ladin, and R.F. Weaver, Physical mapping of
temporally regulated, overlapping transcripts in the region of the 10K
protein gene in Autographa californica nuclear polyhedrosis virus. J.
Virol., 57 (1986) 18-27.
[17] T. Y. Wu, C. Y. Wu, Y. J. Chen, C. Y. Chen, and C. H. Wang, The 5'
untranslated region of Perina nuda virus (PnV) possesses a strong internal
translation activity in baculovirus-infected insect cells. FEBS Lett. 581
(2007) 3120-3126.
@article{"International Journal of Biological, Life and Agricultural Sciences:50916", author = "Tzyy Rong Jinn and Nguyen Tiep Khac and Tzong Yuan Wu", title = "Production of H5N1 Hemagglutinin inTrichoplusia ni Larvae by a Novel Bi-cistronic Baculovirus Expression Vector", abstract = "Highly pathogenic avian influenza (HPAI) H5N1 viruses have created demand for a cost-effective vaccine to prevent a pandemic of the disease. Here, we report that Trichoplusia ni (T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an potential effective vaccine for chickens. To facilitate the recombinant virus identification, virus titer determination and access the infected larvae, we employed the internal ribosome entry site (IRES) derived from Perina nuda virus (PnV, belongs to insect picorna like Iflavirus genus) to construct a bi-cistronic baculovirus expression vector that can express the rH5HA protein and enhanced green fluorescent protein (EGFP) simultaneously. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa H5HA1) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for H5N1 vaccine production.
", keywords = "Avian Influenza, baculovirus, hemagglutinin, Trichoplusia ni larvae", volume = "4", number = "5", pages = "228-4", }
