Baculovirus and Insect Cell Expression Protocols pp 267-279

Part of the Methods in Molecular Biology™ book series (MIMB, volume 388)

Protein Production With Recombinant Baculoviruses in Lepidopteran Larvae

  • Yi Liu
  • Nathan De Carolis
  • Nikolai van Beek


With an increasing need for functional analysis of proteins, there is a growing demand for fast and cost-effective production of biologically active eukaryotic proteins. The baculovirus expression vector system is widely used, and in the vast majority of cases cultured insect cells have been the host of choice. A low cost alternative to bioreactor-based protein production exists in the use of live insect larvae as “mini bioreactors.” In this chapter, we focus on Trichoplusia ni as the host insect for recombinant protein production, and explore three different methods of virus administration to the larvae. The first method is labor-intensive, as extracellular virus is injected into each larva, whereas the second lends itself to infection of large numbers of larvae via oral inoculation. While these first two methods require cultured insect cells for the generation of recombinant virus, the third relies on transfection of larvae with recombinant viral DNA and does not require cultured insect cells as an intermediate stage. We suggest that small-to mid-scale recombinant protein production (mg-g level) can be achieved in T. ni larvae with relative ease.

Key Words

Trichoplusia ni cabbage looper baculovirus recombinant protein expression transfection of insect larvae 


  1. 1.
    Maeda, S., Kawai, T., Obinata, M., et al. (1985) Production of human α-interferon in silkworm using a baculovirus vector. Nature 315, 592–594.PubMedCrossRefGoogle Scholar
  2. 2.
    Miyajima, A., Schreurs, J., Otsu, K., Kondo, A., Arai, K., and Maeda, S. (1987) Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3. Gene 58, 273–281.PubMedCrossRefGoogle Scholar
  3. 3.
    Reis, U., Blum, B., von Specht, B.-U., Domdey, H., and Collins, J. (1992) Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus. Bio/Technology 10, 910–912.PubMedCrossRefGoogle Scholar
  4. 4.
    Muneta, Y., Zhao, H. K., Inumaru, S., and Mori, Y. (2003) Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system. J. Vet. Med. Sci. 65, 219–223.PubMedCrossRefGoogle Scholar
  5. 5.
    Mann, S. J., Lamming, G. E., Fisher, P. A., Maeda, S., and Stewart, H. J. (2002) Production of recombinant ovine interferon τ using a Bombyx mori nuclear polyhedrosis baculovirus expression system. Anim. Biotechnol. 13, 149–158.PubMedCrossRefGoogle Scholar
  6. 6.
    Suzuki, T., Kaki, H., Naya, S., et al. (2002) Recombinant human chymase produced by silkworm-baculovirus expression system: Its application for a chymase detection kit. Jpn. J. Pharmacol. 90, 210–213.PubMedCrossRefGoogle Scholar
  7. 7.
    Nagaya, H., Kanaya, T., Kaki, H., et al. (2004) Establishment of a large-scale purification procedure for purified recombinant bovine interferon-τ produced by a silkworm-baculovirus gene expression system. J. Vet. Med. Sci. 66, 1395–1401.PubMedCrossRefGoogle Scholar
  8. 8.
    Gretch, D. G., Sturley, S. L., Friesen, P. D., Beckage, N. E., and Attie, A. D. (1991) Baculovirus-mediated expression of human apolipoprotein E in Manduca sexta larvae generates particles that bind to the low density lipoprotein receptor. Proc. Natl. Acad. Sci. USA 88, 8530–8533.PubMedCrossRefGoogle Scholar
  9. 9.
    Hellers, M. and Steiner, H. (1992) Diapausing pupae of Hyalophora cecropia: an alternative host for baculovirus mediated expression. Insect Biochem. Molec. Biol. 22, 35–39.CrossRefGoogle Scholar
  10. 10.
    Ahmad, S., Bassiri, M., Banerjee, A., and Yilma, T. (1993) Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larvae: use in differential diagnosis between vaccinated and infected animals. Virology 192, 207–216.PubMedCrossRefGoogle Scholar
  11. 11.
    Kuroda, K., Groener, A., Frese, K., et al. (1989) Synthesis of biologically active influenza virus hemagglutinin in insect larvae. J. Virol. 63, 1677–1685.PubMedGoogle Scholar
  12. 12.
    Richardson, C. D, Banville, M., Lalumiere, M., Vialard, J., and Meighen, E. A. (1992) Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirology 34, 213–227.PubMedGoogle Scholar
  13. 13.
    Argaud, O., Croizier, L., Lopez-Ferber, M., and Croizier, G. (1998) Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae. J. Gen. Virol. 79, 931–935.PubMedGoogle Scholar
  14. 14.
    Maeda, S., Kamita, S. G., and Kondo, A. (1993) Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV. J. Virol. 67, 6234–6238.PubMedGoogle Scholar
  15. 15.
    Groener, A. (1989) Host range of AcNPV, in The Biology of Baculoviruses, vol. 1, (Granados, R. R. and Federici, B. A., eds.), CRC, Boca Raton, FL, pp. 177–188.Google Scholar
  16. 16.
    Villalba, M., Wente, S. R., Russell, D. S., Jongcheol, A., Reichelderfer, C. F., and Rosen, O. M. (1989) Another version of the human insulin receptor kinase do main: Expression, purification, and characterization. Proc. Natl. Acad. Sci. USA 86, 7848–7852.PubMedCrossRefGoogle Scholar
  17. 17.
    Medin, J. A., Hunt, L., Gathy, K., Evans, R. K., and Coleman, M. S. (1990) Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae. Proc. Natl. Acad. Sci. USA 87, 2760–2764.PubMedCrossRefGoogle Scholar
  18. 18.
    Tremblay, N. M., Kennedy, B. P, Street, I. P., Kaupp, W. J., Laliberte, F., and Weech, P. K. (1993) Human group II phospholipase A2 expressed in Trichoplusia ni larvae: isolation and kinetic properties of the enzyme. Protein Expr. Purif. 4, 490–498.PubMedCrossRefGoogle Scholar
  19. 19.
    Pham, M.-Q., Naggie, S., Wier, M., Cha, H. J., and Bentley, W. E. (1999) Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolosis. Biotechnol. Bioeng. 62, 175–182.PubMedCrossRefGoogle Scholar
  20. 20.
    Cha, H. J., Dalal, N. G., Pham, M.-Q., Kramer, S. F., Vakharia, V. N., and Bentley, W. E. (2002) Monitoring foreign protein expression under baculovirus p10 and polh promoters in insect larvae. BioTechniques 32, 986–991.PubMedGoogle Scholar
  21. 21.
    Hale, C. C., Zimmerschied, J. A., Bliler, S., and Price, E. M. (1999) Large-scale expression of recombinant cardiac sodium-calcium exchange in insect larvae. Prot. Expr. Purif. 15, 121–126.CrossRefGoogle Scholar
  22. 22.
    Wood, H. A., Trotter, K. M., Davis, T. R., and Hughes, P. R. (1993) Per os infectivity of preoccluded virions from polyhedrin-minus recombinant baculoviruses. J. Invertebr. Pathol. 62, 64–67.CrossRefGoogle Scholar
  23. 23.
    Wood, H. A. (1997) Stable pre-occluded virus particle. US Patent 5,593,669.Google Scholar
  24. 24.
    Wood, H. A. (2000) Stable pre-occluded virus particle for use in recombinant protein production and pesticides. US Patent 6,090,379.Google Scholar
  25. 25.
    O’Reilly, D. R., Brown, M. R., and Miller, L. K. (1992) Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm, Spodoptera frugiperda: host ecdysteroids are conjugated with galactose. Insect Biochem. Mol. Biol. 22, 313–320.CrossRefGoogle Scholar
  26. 26.
    Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11,984–11,989.PubMedCrossRefGoogle Scholar
  27. 27.
    Wu, X., Cao, C., Kumar, V. S., and Cui, W. (2004) An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin. Res. Microbiol. 155, 462–466.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Yi Liu
    • 1
  • Nathan De Carolis
    • 2
  • Nikolai van Beek
    • 2
  1. 1.BIOSET, Inc.Rockville
  2. 2.Chesapeake PERL, Inc.Savage

Personalised recommendations