Abstract
Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Backovic M, Jardetzky TS (2011) Class III viral membrane fusion proteins. Adv Exp Med Biol 714:91–101. doi:10.1007/978-94-007-0782-5_3
Blissard GW, Rohrmann GF (1989) Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 170:537–555. doi:10.1016/0042-6822(89)90445-5
Blissard GW, Wenz JR (1992) Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66:6829–6835
Boulain JC, Charbit A, Hofnung M (1986) Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12. Mol Gen Genet 205:339–348. doi:10.1007/BF00430448
Fedeson DT, Ducat DC (2016) Cyanobacterial surface display system mediates engineered interspecies and abiotic binding. ACS Synth Biol. doi:10.1021/acssynbio.6b00254
Grabherr R, Ernst W (2010) Baculovirus for eukaryotic protein display. Curr Gene Ther 10:195–200. doi:10.2174/156652310791321297
Ikonomou L, Schneider YJ, Agathos SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62:1–20. doi:10.1007/s00253-003-1223-9
Kadlec J, Loureiro S, Abrescia NG, Stuart DI, Jones IM (2008) The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines. Nat Struct Mol Biol 15:1024–1030. doi:10.1038/nsmb.1484
Khurana S, Fuentes S, Coyle EM, Ravichandran S, Davey RT Jr, Beigel JH (2016) Human antibody repertoire after VSV-Ebola vaccination identifies novel targets and virus-neutralizing IgM antibodies. Nat Med 22:1439–1447. doi:10.1038/nm.4201
Li Z, Blissard GW (2011) Autographa californica multiple nucleopolyhedrovirus GP64 protein: roles of histidine residues in triggering membrane fusion and fusion pore expansion. J Virol 85:12492–12504. doi:10.1128/JVI.05153-11
Mann SG, King LA (1989) Efficient transfection of insect cells with baculovirus DNA using electroporation. J Gen Virol 70:3501–3505. doi:10.1099/0022-1317-70-12-3501
Oomens AG, Blissard GW (1999) Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus. Virology 254:297–314. doi:10.1006/viro.1998.9523
Peralta A, Maroniche GA, Alfonso V, Molinari P, Taboga O (2013) VP1 protein of Foot-and-mouth disease virus (FMDV) impairs baculovirus surface display. Virus Res 175:87–90. doi:10.1016/j.virusres.2013.03.018
Rohrmann GF (2013) Structural proteins of baculovirus occlusion bodies and virions. In: Rohrmann GF (ed) Baculovirus molecular biology. 3rd edn. Bethesda, Maryland, pp 23–46
Roy A, Lu CF, Marykwas DL, Lipke PN, Kurjan J (1991) The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin. Mol Cell Biol 11:4196–4206. doi:10.1128/MCB.11.8.4196
Santos NGL, Rocca MP, Pereira CA, Ventini DC, Puglia AP, Jorge SAC, Lemos MAN, Astray RM (2016) Impact of recombinant Drosophila S2 cell population enrichment on expression of rabies virus glycoprotein. Cytotechnology 68:2605–2611. doi:10.1007/s10616-016-9984-z
Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317. doi:10.1126/science.4001944
Tang M, Sun X, Zhang S, Wan J, Li L, Ni H (2017) Improved catalytic and antifungal activities of Bacillus thuringiensis cells with surface display of Chi9602DeltaSP. J Appl Microbiol 122:106–118. doi:10.1111/jam.13333
Xu XG, Wang ZS, Zhang Q, Li ZC, Zhao HN, Li W, Tong DW, Liu HJ (2011) Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models. Vaccine 29:636–643. doi:10.1016/j.vaccine.2010.11.045
Zhao SL, Dai XJ, Liang JS, Liang CY (2012) Surface display of rice stripe virus NSvc2 and analysis of its membrane fusion activity. Virol Sin 27:100–108. doi:10.1007/s12250-012-3237-x
Zhou J, Blissard GW (2008) Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus. J Virol 82:4449–4460. doi:10.1128/JVI.02490-07
Acknowledgements
This study was supported by National Natural Science Foundation of China (31272376), and Key Research and Development Program in Shandong Province (2015GNC111003).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Qi, BX., Chen, YJ., Su, R. et al. Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic. Cytotechnology 69, 775–783 (2017). https://doi.org/10.1007/s10616-017-0086-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-017-0086-3