Abstract
Baculovirus has been widely used for the production of numerous recombinant proteins in insect cells. Baculovirus vectors have several advantages, including proper post-translational modification, biosafety, and multiple large gene expression ability. Most insect cell-produced proteins have been expressed by using the baculovirus expression vector system (BEVS) under the control of strong polyhedrin (Polh) or p10 promoters. There has been no report on the expression of recombinant proteins by baculovirus in plant cells. In this study, we used the baculovirus vector to express recombinant green fluorescent protein (GFP) in plants. To investigate the expression of GFP protein by baculovirus in plants, we cloned the gfp gene under the control of Polh promoter or Cauliflower Mosaic Virus (CaMV) 35S promoter to yield the Polh-GFP and 35S- GFP bacmids carrying the GFP expression cassettes, respectively. The presence of Polh-GFP and 35S-GFP expression cassettes in the bacmids was confirmed by polymerase chain reaction (PCR). Subsequently, both the GFP bacmids and GFP baculovirus vectors generated from the bacmid-transfected Sf9 insect cells were inoculated into Nicotiana benthamiana leaves. Confocal microscopy revealed that the gfp gene expression was high in plant leaves at 48 and 72 h after bacmid and baculovirus inoculation. Reverse transcription-PCR (RT-PCR) and fluorescence microscopy confirmed that the gfp genes under the control of Polh or CaMV35S promoters were highly expressed in plant leaves inoculated with 40 L of baculovirus solution. These results suggested that the baculovirus vector can be used to express recombinant proteins in plants. The baculovirus vector- mediated gene delivery and expression system could be used in plant biotechnology for fast and efficient production of recombinant proteins and for molecular virology studies in plants.
Similar content being viewed by others
Literature Cited
Andreason, G.L. and G.A. Evans. 1988. Introduction and expression of DNA molecules in eukaryotic cells by electroporation. Biotechniques 6:650–660.
Beljelarskaya, S.N. 2002. A Baculovirus expression system for insect cells. Mol. Biol. 36:281–292.
Bishop, D.H.L., P.F. Entwistle, I.R. Cameron, C.J. Allen, and R.D. Possee. 1988. In: M. Sussman, C.H. Collins, F.A. Skinner, D.E. Stewart- Tull (eds.). Field trials of genetically-engineered baculovirus insecticides. The Release of Genetically-engineered Micro-organisms, p. 143–179. Academic Press Limited, London, UK.
Boyce, F.M. and N.L.R. Bucher. 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl. Acad. Sci. USA. 93:2348–2352.
Cameron, I.R. and D.H.L. Bishop. 1989. Insect cell culture technology in baculovirus expression system. Trends Biotechnol. 7:66–70.
Carbonell, L.F., M.J. Klowen, and L.K. Miller. 1985. Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J. Virol. 56:153–160.
Cemazar, M., G. Sersa, J. Wilson, G.M. Tozer, S.L. Hart, A. Grosel, and G.U. Dachs. 2002. Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, liposomes and integrin-targeted vector. Cancer Gene Ther. 9:399–406.
Cory, J.S. and D.H. Bishop. 1997. Use of baculoviruses as biological insecticides. Mol. Biotechnol. 7:303–313.
Darbani, B., A. Eimanifar, C.N. Stewart, and W.N. Camargo. 2008. DNA delivery methods to produce transgenic plants. Turk. J. Biol. 7:385–402.
Fassler, J.S. and G.N. Gussin. 1996. Promoters and basal transcription machinery in eubacteria and eukaryotes: concepts, definitions, and analogies. Meth. Enzymol. 273:3–29.
Felgner, P.L., T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, G.M. Ringold, and M. Danielsen. 1987. Lipofection: highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA. 84:7413–7417.
Fujita, R., T. Matsuyama, J. Yamagishi, K. Sahara, S. Asano, and H. Bando. 2006. Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and upregulation of the host beta-actin gene. J. Virol. 80:2390–2395.
Gao, H., Y. Wang, N. Li, W.P. Peng, Y. Sun, G.Z. Tong, and H.J. Qiu. 2007. Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells. J. Biotechnol. 131:138–143.
Hill, D.R., J.J. Aumiller, X.Z. Shi, and D.L. Jarvis. 2006. Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT-1 cells cultured in serum-free medium. Biotechnol. Bioeng. 95:37–47.
Hofmann, C., V. Sandig, G. Jennings, M. Rudolph, P. Schlag, and M. Strauss. 1995. Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc. Natl. Acad. Sci. USA. 92:10099–10103.
Jarvis, D.L. 2003. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virol. 310:1–7.
Jarvis, D.L. and E.E. Finn. 1996. Midifying the inset cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat. Biotechnol. 14:1288–1292.
Kenoutis, C., R.C. Efrose, L. Swevers, A.A. Lavdas, M. Gaitanou, R. Matsas, and K. Iatrou. 2006. Baculovirus-mediated gene delivery into mammalian cells does not alter their transcriptional and differentiating potential but is accompanied by early viral gene expression. J. Virol. 80:4135–4146.
King, L.A. and R.D. Possee. 1992. The baculovirus expression system: A laboratory manual. p. 1–347. Chapman and Hall, New York, USA.
Kost, T.A. and J.P. Condreay. 2002. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol. 20:173–180.
Luckow, V.A. and M.D. Summers. 1988. Trends in the development of baculovirus expression vectors. Biotechnol. 6:47–55.
Matsuura, Y., R.D. Possee, H.A. Overton, and D.H.L. Bishop. 1987. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J. Gen. Virol. 68: 1233–1250.
Miller, L.K. 1988. Baculoviruses as gene expression vectors. Annu. Rev. Microbiol. 42:177–199.
Nishikawa, M. and L. Huang. 2001. Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum. Gene Ther. 12:861–870.
Ohashi, S., T. Kubo, T. Ikeda, Y. Arai, K. Takahashi, Y. Hirasawa, M. Takigawa, E. Satoh, J. Imanishi, and O. Mazda. 2001. Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma- derived HCS-2/8 cells. J. Orthop. Sci. 6:75–81.
O’Reilly, D.R., L.K. Miller, and V.A. Luckow. 1992. Baculovirus expression vectors: A Laboratory Manual, W.H. Freeman and Company, New York, USA.
Patel, G., K. Nasmyth, and N. Jones. 1992. A new method for the isolation of recombinant baculovirus. Nucl. Acids Res. 20:7–104.
Pennock, G.D., C. Shoemaker, and L.K. Miller. 1984. Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol. Cell Biol. 4:399–406.
Philipps, B., M. Forstner, and L.M. Mayr. 2005. A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells. Biotechnol. Prog. 21:708–711.
Possee, D.R. 1997. Baculovirus as expression vectors. Cur. Opin. Biotechnol. 8:569–572.
Rose, J.K. 1996. Transfection of DNA into eukaryotic cells - Optimization of transfection, p. 9.1.1–9.1.4. In: F.M. Ausubel (ed.). Curr. Protoc. Mol. Biol., John Wiley & Sons. Inc., New York, USA.
Selden, R.F. 1996. Transfection using DEAE-dextran. p. 9.2.1–9.2.4. In: F.M. Ausubel (ed.). Curr. Protoc. Mol Biol. John Wiley & Sons.Inc., New York, USA.
Smith, G.E., M.D. Summers, and M.J. Fraser. 1983. Production of human -interferon in insect cells infected with a baculovirus expression vector. Mol. Cell Biol. 3:2156–2165.
Summers, M.D. and G.E. Smith. 1987. A manual of methods for baculovirus vectors and insect cell culture procedure. Tx Ag Expt Stn Bull No. 1555. College Station, TX, USA.
Van Loo, N.D., E. Fortunati, E. Ehlert, M. Rabelink, F. Grosveld, and B.J. Scholte. 2001. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids. J. Virol. 75:961–970.
Watanabe, S.Y., A.M. Albsoul-Younes, T. Kawano, H. Itoh, Y. Kaziro, S. Nakajima, and Y. Nakajima. 1999. Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology. Neurosci. Res. 33:71–78.
Zhang, Y.H., G. Enden, and J.C. Merchuk. 2005. Insect cells- Baculovirus system: Factors affecting growth and low MOI infection. Biochem. Eng. J. 27:8–16.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to this work.
Rights and permissions
About this article
Cite this article
Oh, EY., Kim, Y.K., Park, DY. et al. Expression of recombinant proteins in plants by using baculovirus vectors. Hortic. Environ. Biotechnol. 52, 95–104 (2011). https://doi.org/10.1007/s13580-011-0158-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13580-011-0158-9