Introduction

Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is a typical encapsulated double-stranded DNA virus. It is capable of transducing non-dividing cells (van Loo et al. 2001) and has low cytotoxicity in mammalian cells even at a very high virus load (Yap et al. 1997; Sandig et al. 1996; Shoji et al. 1997). Due to its characteristic advantages of security, economy and convenience, the baculovirus display system (BDS) is regarded as the most versatile surface display system in eukaryotes (Hulst et al. 1994; Chen et al. 2007). The construction of BDS involves fusing the target protein with the type I or III envelope proteins of baculovirus, thus realizing the display of the target protein on the surface of the recombinant baculovirus envelope (Lin et al. 2008; Whitford et al. 1989; Yang et al. 2007). This method has been extended to develop the surface display of target proteins for eukaryotes. It has become a construction platform for gene therapy and genetic engineering vaccine with important application potential (Xu et al. 2008; Xu and Liu 2008; Lin et al. 2008; Tami et al. 2004; Yoshida et al. 2003).

A wide range of applications have been proposed since baculovirus was found to transduce into mammalian cells (Hu 2005). The attachment and endocytosis of baculovirus into insect cells are mediated by an envelope glycoprotein GP64. The mature structure of GP64 includes a transmembrane domain (TM) and cytoplasmic domain (CTD) (Monsma et al. 1996; Kitagawa et al. 2005). In addition, a signal peptide (sp) was located in the N-terminal of mature GP64 and directs its transport to the plasma membrane after expression in cells, where GP64 appears as homotrimers on the surface of infected cells. The CTD domain is responsible for contact with the nucleocapsid of baculovirus and guides its encapsulation by the envelope (Yang et al. 2007; Oomens and Blissard 1999). The significance of GP64 in virus budding has been used to enact the surface display of target peptides by inserting a heterologous peptide between the sp and mature domain of GP64 (O’Reilly 1997). After expression along with wild-type GP64, fusion GP64 is translocated to the cell membrane and assembled into the baculovirus envelope. In addition to GP64, fusions can be made to the N-terminus or C terminus of the major capsid protein VP39 without compromising the viral titer or functionality (Yoshida et al. 2003). Another strategy developed non-polar protein distribution on the baculovirus envelope, consisting of the expression of foreign peptide inserted between sp of GP64 and vesicular stomatitis virus (VSV) G protein (Peralta et al. 2013; Oker-Blom et al. 2003; Chapple and Jones 2002).

Since the GP64 fusion protein competes strongly with the wild-type GP64 envelope protein expressed by baculovirus, it leads to a low-efficiency of target protein display on the viral surface. The main objective of this study was to construct the envelope glycoprotein GP64 gene auxotrophic strain E.coli SW106 Bacmid, in the hope that internal ribosome entry site (ires)-dependent low-expression of wild-type gp64 (Mountford and Smith 1995; Pelletier and Sonenberg 1988) would improve the display efficiency of the target protein without affecting the baculovirus replication cycle. Here, we constructed a recombinant baculovirus AcMNPV-Δgp64-G64-M64-Y64-I64 with the ability of high display efficiency and multi-gene co-display.

Materials and methods

Bacterial strains, plasmids, viral Bacmid, reagents and insect cell

E.coli DH10B, BW23474, and TOP10 are used for the propagation of Bacmid, R6kγ origin-derived plasmids, and other general plasmids, respectively. E.coli SW106 Bacmid containing Bacmid, pHelper, and pGB2Ωinv were constructed previously (Yao et al. 2012, 2010). pUCDM and pFBDM were from Prof. Richmond (Berger et al. 2004). pFBDM and pUCDM, as well as the modified Bacmid with gentamycin or chloramphenicol resistance gene by mini-Tn7 or cre-loxp transposition, were from our previous study (Sun et al. 2009; Yao et al. 2007). pBac-IR-eGFP containing the 59-UTR internal ribosome entry site (ires) sequence was provided by Prof. Wu (Motohashi et al. 2005). pIZTV5 was purchased from Invitrogen.

Pfu Taq, restriction enzymes, and T4 DNA ligase were purchased from NEB (New England Biolabs, England), while DL-α-ε Diaminopimelic acid (DAP) was bought from Sigma (cat.D1377, USA). Low salt (LS) medium (10 g of tryptone, 5 g of NaCl and 5 g of yeast extract in 1 L of broth, pH7.5) was used for cloning and growing the plasmids containing zeocin resistance gene. Spodoptera frugiperda 9 (Sf9) cells were maintained at 27 °C in Grace medium supplemented with 10% FBS (Gibco).

Generation of the envelope glycoprotein gp64 gene auxotrophic strain E.coli SW106 Bacmid

The genomic DNA of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was extracted using a genomic DNA extraction kit (TaKaRa). Deletion of gp64 gene results in a failure of budding and invasion by baculovirus. The gp64 gene homologous recombinant arms contain gp64F (located upstream 400 bp of the promoter p64) and gp64R (located downstream 400 bp of p64), which were obtained by polymerase chain reaction (PCR) from AcMNPV DNA (Table 1). The zeocin resistance cassette was amplified with primer Zeo-F and Zeo-R (Table 1) from pIZTV5, which was inserted between gp64F and gp64R by over-lap PCR. The homologous recombination fragment (gp64F-Zeo-gp64R) was verified by sequencing and electroporated into the 42 °C-induced electro-competent E.coli SW106 Bacmid prepared according to the previous work (Warming et al. 2005). The transformed cells were spread on LS plate supplemented with 50 µg/mL kanamycin, 10 µg/mL spectinomycin, 10 µg/mL tetracycline, 25 µg/mL zeocin, 0.5 mM DAP, and cultured at 32 °C overnight. The positive colonies grown on the kan/Spe/Tet/Zeo/DAP plate were picked and identified by PCR (primer gp64F-F and gp64R-R, Table 1). The zeocin cassette was then removed from the Bacmid by Flp-mediated excision (Cherepanov and Wackernagel 1995). The resulting Bacmid gp64 auxotrophic strain was named E.coli SW106 Bacmid-Δgp64.

Table 1 Primers used in the study
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Construction of donor vectors

The coding sequence for egfp was amplified by PCR using primer egfp-F and egfp-R (Table 1). To follow transfection and infection processes, the PCR product was digested with BamH I and Sac I, and ligated to the downstream of the promoter polyhedrin (polh) on the pFBDM, forming a donor vector pF-polh-G (Fig. 1a). Primers ires-F and ires-R (Table 1) were used to amplify the ires gene by pBac-IR-eGFP, which was ligated into the Sac I and Xba I of pF-polh-G. The N-terminal of gp64 gene fusing with sp was amplified from AcMNPV DNA by PCR using spgp64-F and spgp64-R. The PCR product was ligated to the downstream of the ires by Xba I and Pst I, to form another vector pF-polh-G-I64 (Fig. 1b), in which the spgp64 was dependent by ires element.

Fig. 1
figure 1

The gene arrangement on recombinant donor vectors. a The map of pF-polh-G shows an expression cassette including promoter polh and egfp. b The map of pF-polh-G-I64 shows an expression cassette including promoter polh, egfp, and ires-dependent gp64 expression cassette iresspgp64. c The maps of pF-p64sp-R64-polhF-polh-G show two expression cassettes including egfp and Firefly luciferase are driven by polh, and a fusion gene fragment of sp, Renilla luciferaseΔTAA and gp64. d The maps of pF-p64sp-R64-polhF-polh-G-I64 show two expression cassettes including egfp, iresspgp64, and Firefly luciferase are driven by polh, and a fusion gene fragment of sp, Renilla luciferaseΔTAA and gp64. e The maps of pU-p64spH-G64-I64 show a fusion gene fragment of sp, 6 × His-tag, egfpΔTAA, gp64 and iresspgp64. f The map of pF-p64spF-M64-p64spS-Y64 show two fusion gene fragments of sp, strep II-tag, Flag-tag, eyfpΔTAA, mCherryΔTAA, gp64

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The primers Fluc-F and Fluc-R were used to amplify the Firefly luciferase (Fluc) gene (Table 1), and were cloned into the downstream of the polh on the pFBDM by the BamH I and Sal I sites to generate the vector pF-polh-F. The C-terminal of promoter p64 fusing with sp (p64sp) was amplified from AcMNPV DNA by PCR using p64sp-F and p64sp-R. The PCR product was digested with Spe I and Xma I, and then ligated to the same sites of pF-polh-F to replace promoter p10. The flexible linker-peptide sequence (GGGGSGGGGSGGGGS) was fused to the C terminus of Renilla luciferase ΔTAA (deletion of termination codon, Rluc(G4S)3ΔTAA), and cloned under p64sp. The gp64 was amplified from AcMNPV DNA by PCR using primer gp64-F and gp64-R, and then was ligated under the Rluc(G4S)3ΔTAA to generate the transient vector pF-p64sp-R64-polh-F (Table 1). The pF-polh-G was digested with Cla I / Avr II to release DNA polh-egfp, and the fragment was cloned into pF-p64sp-R64-polh-F via Cla I and Spe I (Avr II and Spe I were isocaudamer) to construct donor vector pF-p64sp-R64-polh-F-polh-G (Fig. 1c). Similarly, the donor vector pF-p64sp-R64-polh-F-polh-G-I64 (Fig. 1d) was successfully constructed by pF-p64sp-R64-polh-F and pF-polh-G-I64.

The C-terminal of p64 containing sp and different label proteins (6 × His/ Sterp II/ Flag tag) were amplified from AcMNPV DNA by PCR to form three kinds of PCR products: p64spHis (6 × His-tag; sequence: 5′-CATCATCACCACCATCAC), p64spSterp II (Sterp II-tag; sequence: 5′-TGGAGCCACCCGCAGTTTGAAAAG), and p64spFlag (Flag-tag; sequence: 5′-GATTACAAGGATGACGACGATAAG), respectively. The p64spHis was ligated into pUCDM to replace polh via Cla I and BamH I. The egfpΔTAA was amplified from pBac-IR-eGFP using primers egfpΔTAA-F and egfpΔTAA-R and ligated into the BamH I and Sal I under p64spHis. gp64 was amplified from AcMNPV DNA using primers gp64-F and gp64-R and ligated into Sal I and Sac I under the p64spHis to generate the conditional replication transposition transient vector pU-p64spH-G64. pF-polh-G-I64 was digested with Sac I and Pst I to release DNA iresspgp64. The fragment was then cloned into the same sites of pU-p64spH-G64 to make donor vector pU-p64spH-G64-I64 (Fig. 1e).

To replace polh and p10, PCR products p64spSterp II and p64spFlag were respectively cloned into pFBDM through Cla I / BamH I and Spe I / Xma I, forming another transient vector pF-p64spF-p64spS. The eyfpΔTAA and mCherryΔTAA were amplified using primers eyfpΔTAA-F/eyfpΔTAA-R and mCherryΔTAA-F/mCherryΔTAA-R, then cloned into pF-p64spF-p64spS via BamH I/Sal I and Xma I / Xho I under the p64spSterp II and p64spFlag. The gp64 fragment was amplified from AcMNPV and successively introduced by Xho I/Sph I and Sal I/Sac I to construct donor vector pF-p64spF-M64-p64spS-Y64 (Fig. 1f).

Introduction of multiple genes into Bacmid

Donor vectors pF-polh-G, pF-polh-G-I64, and pF-p64sp-R64-polh-F-I64 were transferred into E.coli SW106 Bacmid-Δgp64 through mini-Tn7 transposition (Sun et al. 2009; Yao et al. 2007) to generate three kinds of Bacmids, including Bacmid-Δgp64-G, Bacmid-Δgp64-G-I64, and Bacmid-Δgp64-p64sp-R64-polh-F-I64, respectively. Similarly, pF-polh-G and pF-p64sp-R64-polh-F were separately transferred into E.coli SW106 Bacmid to construct two other kinds of recombinant Bacmid-G and Bacmid-p64sp-R64-polh-F as control. Furthermore, egfp, eyfp, and mCherry were introduced from pU-p64spH-G64-I64 and pF-p64spF-M64-p64spS-Y64 into E.coli SW106 Bacmid-Δgp64 by cre-loxp and mini-Tn7 site-specific recombination to construct the recombinant Bacmid-Δgp64-p64sp-HG-FM-SY-I64. The positive recombinant Bacmid was identified by white–blue and PCR screening as per the previous report (Yao et al. 2012, 2010; Berger et al. 2004).

Production of recombinant baculoviruses

E. coli SW106 cells with different recombined foreign genes were cultured until OD600 = 0.5–1 (attendance at 600 nm). These cells were collected by centrifugation (3000g) and resuspended in serum-free insect medium. The bacterial suspension was adjusted to different densities (105–108 cells/mL) with serum-free Grace’s insect medium (Yao et al. 2012, 2010). Insect cells sf9 were cultured overnight in a 24-well plate until the cell density was approximately 70–80%. The supernatant was discarded and different concentrations of bacteria were added to the corresponding wells. After culturing at 28 °C for 4–5 h, bacteria in each well was washed out by serum-free Grace’s insect medium. 500 µL of fresh insect medium (with 10% FBS and 0.075% of penicillin) was then added and incubated for 4–5 d.p.i. When the fluorescence in the corresponding well was observed by fluorescence microscope, indicating that sf9 cells were infected successfully, the supernatant was collected and infected again with sf9 cells. Fluorescence appeared again at 4–5 d.p.i, indicating that the recombinant baculovirus was successfully constructed and distributed in the cell supernatant. In this study, the purified Bacmids were respectively transfected into Sf9 cells to produce six kinds of recombinant baculoviruses, which we named AcMNPV-polhG, AcMNPV-Δgp64-polhG, AcMNPV-Δgp64-polhGI64, AcMNPV-Δgp64-p64spR64-polhF, AcMNPV-Δgp64-p64spR64-polhFI64, and AcMNPV-Δgp64-p64sp-HG-FM-SY-I64. The plaque assay technique was used to determine the recombinant virus titer (Roldao et al. 2009).

Dual-Glo luciferase assay system

To prevent host cell apoptosis and break down, infected sf9 cells were collected by low-speed centrifugation (500g) and washed with PBS at 72 h.p.i. The expression of Renilla luciferase on the surface of the whole cell membrane was measured using the Dual-Glo luciferase assay system (Cat NO.E1910, Promega). The termination solution and cell lysate was added, breaking the cell, after which the intracellular Firefly luciferase was released and determined.

Baculovirus purification and titer determination

The supernatant of sf9 cells infected with recombinant baculovirus (MOI = 1) was collected at 4 d.p.i. Baculovirus in the supernatant was purified by two rounds of sucrose gradient ultracentrifugation according to standard methods (O’Reilly 1997). The baculovirus titers were measured by the TCID50 method according to standard methods.

SDS–PAGE and western blot

The purified baculoviruses or infected cell lysates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Three primary antibodies including 6 × His / Sterp II/Flag tag (1:4000, Beyotime) were used to detect fusion protein in the western blot. The secondary antibodies were goat anti-porcine and goat anti-mouse IgG conjugated to HRP (1:3000 dilution, Invitrogen). The protein bands were visualized by the ECL chemiluminescence system Hyper-Max films, as recommended by the manufacturer.

Laser scanning confocal microscopy and transmission electron microscope

The sf9 cells were cultured on sterile cover slips (placed in 6-well plates) and infected with baculoviruses at MOI = 10. At 72 h.p.i, infected sf9 cells were mounted on glass slides in 50% glycerol and examined under with confocal laser scanning microscopy (CLSM).

Purified baculoviruses were adsorbed onto glow discharge-activated carbon-coated grids for 2 min. And then, the sample-coated grids were washed three times with distilled water, following a negative staining with 1% uranyl acetate for 45 s. Images were acquired using the JEM 100-CXII transmission electron microscope (TEM).

Results

Construction of gp64-null Bacmid

To improve the display efficiency of the baculovirus, the full gp64 gene was deleted. The homologous recombination fragment gp64F-Zeo-gp64R (Fig. 2a1) was electrotransformed into E.coli SW106 Bacmid (Fig. 2a2). Upon the completion of homologous recombination, wild-type gp64 ORF and p64 (Fig. 2a3) were replaced by a zeocin resistance cassette (Fig. 2b1).

Fig. 2
figure 2

Construction of gp64-null Bacmid. a1 The homologous recombination gene fragment gp64F-Zeo-gp64R; a2 E.coli SW106 Bacmid; a3 wild-type gp64 ORF with promoter p64. b1 gp64 and p64 were replaced by a zeocin resistance cassette. b2 E.coli SW106 Bacmid-Δgp64. c Identification the gp64 ORF and p64 were replaced successfully by PCR with primer gp64F-F / gp64R-R M: DL5000 Marker, 1–4: E.coli SW106-Bacmid-Δgp64; 5: E.coli SW106-Bacmid as control

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The positive colony was further verified by PCR using primers gp64F-F and gp64R-R to amplify of 1,269 kb gp64F-Zeo-gp64R. However, a 1660 kb wild-type gene was amplified from the control with the same primers (Fig. 2c). The gp64-deficient strain was named E.coli SW106 Bacmid-Δgp64.

gp64-null baculovirus was rescued using ires-dependent gp64 expression cassette

No infectious baculovirus was produced when using E.coli SW106 Bacmid-Δgp64 to infect sf9 cell, which proved GP64 was removed as expected (data not show). The extremely late promoter polyhedron (polh) was used to drive egfp, ires, and gp64. The ires-dependent GP64 was used to rescue the infectivity of gp64-null Bacmid. E.coli SW106 Bacmid-Δgp64-G-I64 was successfully constructed (Fig. 3a, b) and used to directly infect sf9 cells (Fig. 3c, d) for the production of recombinant baculovirus (Fig. 3e, f, g). Similarly, E.coli SW106 Ac-G containing the original wild-type gp64 was used as control (Fig. 3h).

Fig. 3
figure 3

gp64-null baculovirus was rescued using ires-dependent gp64 expression cassette. a Donor vector pF-polh-G-I64. b E.coli SW106 Bacmid-Δgp64. c E.coli SW106 Bacmid-Δgp64-G-I64. d sf9 insect cell. e E.coli SW106 Bacmid-Δgp64-G-I64 was unable to synthesize cell walls in sf9 cells, thus rupturing and releasing Bacmid-Δgp64-G-I64. f Generation of recombinant baculovirus AcMNPV-Δgp64-G-I64. g Observation of insect sf9 cells infected by E.coli SW106 Bacmid-Δgp64-G-I64 under Nikon TS100 at 48 h.p.i (g1), 72 h.p.i (g2), 96 h.p.i (g3) and 120 h.p.i (g4), Bar: 200 µm. g5 Purified recombinant baculoviruses (AcMNPV-Δgp64-polh-G-I64) were observed by TEM, Bar: 0.2 µm. Observation of insect sf9 cells infected by AcMNPV-Δgp64-G-I64 under Nikon TS100 at 24 h.p.i (g6), 48 h.p.i (g7), 72 h.p.i (g8) and 96 h.p.i (g9), Bar: 200 µm. h Using E.coli Sw106 Ac-G and AcMNPV-polh-G to perform the same treatment with Group g (as control)

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48 h.p.i later, sf9 cells infected with E.coli SW106 Bacmid-Δgp64-G-I64 began to turn green (Fig. 3g1), which indicated that sf9 cells were infected successfully. Within 48 to 120 h.p.i (Figs. 3g1, 4), the number of green cells gradually increased, and the amount of fluorescence reached a maximum at 120 h.p.i (Figs. 3g, 4), indicating that the baculoviruses were propagating. The culture supernatant was collected and centrifuged at 80000g, after which the pellet was observed with TEM, and a number of mature baculovirus particles AcMNPV-Δgp64-G-I64 were successfully observed (Fig. 3, g5). When the viruses were collected and re-infected in sf9 cells (MOI = 1), green cells were observed at 24 h.p.i (Fig. 3, g5). Similar to the first generation strain’s infection, fluorescence was observed to gradually increase within 24–96 h.p.i and reached a maximum at 96 h.p.i (Figs. 3g, 5g). This shows that recombinant baculoviruses undergo continuous production. Compared with the control (Fig. 3h), it was determined that the low expression of ires-dependent wild-type gp64 had no significant effect on the replication cycle of recombinant baculovirus. This method could be used to improve target protein display efficiency by decreasing the expression of wild-type gp64 by inserting the recombinant virus.

Improvement of target protein display efficiency on the cell surface

Since the AcMNPV envelope membrane is extremely unstable, it is prone to breakage under unfavorable pH or metal ion concentration conditions. Thus, it was difficult to directly observe or account for integral proteins in the baculovirus envelope. To more conveniently and accurately determine the display efficiency of the target protein, we took advantage of the fact that baculovirus acquires the totality of its envelope from the host cell membrane during budding (Fig. 4a). Therefore, the display efficiency of the target protein on the surface of the host cell membrane is a direct reflection of that on the virus capsule.

Fig. 4
figure 4

Improvement of display efficiency of target protein on cell surface. a The GP64 protein fused with Renilla luciferase was displayed on the surface of the sf9 cell membrane. The baculovirus AcMNPV-Δgp64-R64-F-G-I64 completely acquires the host cell membrane as the envelope when budding, resulting in the fusion protein being displayed on the envelope. b Infected 72 h.p.i by AcMNPV-Δgp64-R64-F-G-I64, the sf9 cells were observed by fluorescence microscopy (Bar: 50 µm). c The Renilla luciferase display efficiency (c1) was increased by fourfold compared to control (***p < 0.01)

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The extreme early promoter p64 fused with sp and was used to drive the Renilla luciferase and gp64 fusion protein. Firefly luciferase, egfp and ires-dependent wild-type gp64 were driven by polh promoter. At 72 h.p.i., sf9 cells infected with AcMNPV-Δgp64-R64-F-G-I64 turned green, indicating the successful infection of sf9 cells and the expression of foreign genes (Fig. 4b). GP64 protein fused with Renilla luciferase was displayed on the surface of the cell membrane, while Firefly luciferase was expressed and distributed in the cell. Similarly, baculovirus AcMNPV-R64-F-G was used as a control.

The dual-Glo luciferase assay results showed that the display efficiency of Renilla luciferase on cell surface infected with baculovirus AcMNPV-Δgp64-R64-F-G-I64 is fourfold greater than that of AcMNPV-Δgp64-R64-F-G (Fig. 4c). The assay also showed that ires-dependent gp64 used to rescue wild-type gp64-null Bacmid can significantly increase the display efficiency of the target protein.

Displaying multiple target proteins simultaneously on baculovirus particles

Three different tags 6 × His, Strep II, and Flag were fused with the N terminus of fluorescent proteins eGFP, eYFP, and mCherry, respectively. They were then fused with gp64 N-terminal to construct vectors pU-p64spH-G64-I64 (Fig. 5a) and pF-p64spF-M64-p64spS-Y64 (Fig. 5d). Recombinant baculovirus AcMNPV-Δgp64-G64-M64-Y64-I64, which expressed three kinds of fluorescent proteins fused with GP64, was constructed successfully (Fig. 5f, g).

Fig. 5
figure 5

Diagrammatic sketch for recombinant Bacmid-Δgp64-G64-M64-Y64-I64. a Donor vector pU-p64spH-G64-I64; b E.coli SW106 Bacmid-Δgp64; c E.coli SW106 Bacmid-Δgp64-G64-I64. d Donor vector pF-p64spF-M64-p64spS-Y64. e E.coli SW106 Bacmid-Δgp64-G64-M64-Y64-I64. f The E.coli SW106 Bacmid-Δgp64-G64-M64-Y64-I64 was used to infect the sf9 cells; g recombinant baculovirus AcMNPV-Δgp64-p64sp-HG-FM-SY-I64

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To determine if eGFP, mCherry, and eYFP proteins were properly translocated to the cell surface, sf9 cells were cultured on sterile cover slips, infected at an MOI of 10 by AcMNPV-Δgp64-G64-M64-Y64-I64, and subjected to LSCM at 72 h.p.i. Three fluorescing colors, green, yellow, and red, corresponding to fluorescent proteins eGFP, eYFP, and mCherry were observed along the perimeter of the sf9 cells (Fig. 6a, b, c, d, e), indicating that eGFP, eYFP, mCherry fluorescent proteins were expressed and displayed on the cell membrane as expected. AcMNPV-Δgp64-G64-M64-Y64-I64 virus particles were then collected and purified, and western blot analysis results showed that the three fusion proteins were successfully and simultaneously inducted into the apical membrane of baculovirus (Fig. 6f). Furthermore, Z-section slices and measurements conducted by LSCM revealed that fluorescent proteins were correctly localized on the Sf9 plasma membrane, as opposed to remaining within intracellular structures. (Fig. 6g, h, i, j, k).

Fig. 6
figure 6

Sufficient and stable system of multigene co-display. 72 h.p.i., sf9 cells infected with AcMNPV-Δgp64-G64-M64-Y64-I64 (MOI = 10) were observed using CLSM. a green fluorescence. b red fluorescence; c: yellow fluorescence; d bright; e merge (a + b + c + d). f Purified AcMNPV-Δgp64-G64-M64-Y64-I64 by Western blot, reaction of fusion proteins with 3′ tag-antibodies 6 × His, Sterp II and Flag. Z-section slices and measurements conducted by CLSM showed that fluorescent proteins were correctly localized onto the Sf9 plasma membrane. g bright; h green; i red; j yellow; k merge (g + h + i + j), bar: 10 µm. (Color figure online)

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Discussion

The baculovirus multigene display system (BMDS) is currently the most widely used eukaryotic display system (Boublik et al. 1995). Compared with other display systems, the system has a more complete protein folding and modification mechanism. It has a large capacity (greater than 50 kb) to accept foreign DNA fragments and allows fast and facile construction of high-titer recombinant viruses (Cheshenko et al. 2001; O’Reilly 1997; Davies 1994). Through the advancement of existing recombinant construction methods with novel strategies, BMDS is proving to be a powerful and efficient, yet low-cost method of constructing recombinant baculoviruses. BMDS successfully avoids complex operations such as the extraction of baculovirus genomes and liposome transfection (Yao et al. 2007, 2010, 2012), and exhibits a high degree of efficiency in co-presenting multiple target proteins, making the system an attractive new tool for receptor screening, gene therapy, and genetically engineering vaccines.

Since ires-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines (Mountford and Smith 1995; Pelletier and Sonenberg 1988). The expression of the ires-dependent second gene ranged from 6 to 100% (though in most cases between 20 and 50%) that of the first gene (Mizuguchi et al. 2000; Urabe et al. 1997; Adam et al. 1991). Thus, a single ires-dependent promoter can greatly reduce the competition from wild-type gp64 (Ghattas et al. 1991; Jang et al. 1989, 1988). The results also confirmed that the low-expression of GP64 did not significantly affect the replication cycle of baculovirus. Taken together, this method holds promising prospect in improving the display efficiency of target proteins. In addition, we found that p64 has high activity in early stages after initial virus infection through an analysis of the difference in activity of the baculovirus promoters. Namely its activity is about 20 times that of polh at 12 h.p.i. (data not shown). As such, the display efficiency of target protein can also be improved by p64 to some degree.

Web-based protein functional and structural prediction servers PROSITE (https://prosite.expasy.org/) and PredictProtein (https://open.predictprotein.org/) indicate a linker sequence (GGGGS)3 was added to N terminus of Renilla luciferase to provide distance and flexibility for the N-terminal fusion proteins to fold correctly (Kukkonen et al. 2003). The results also showed that the Renilla luciferase still had a very strong enzyme activity, indicating that the system did not affect the activity of the target protein significantly while still displaying the target protein efficiently.

Our development of BMDS significantly improves the limitations of classical BDS, which lacks a donor vector system and mature technology, and is focused on the display of a single target protein or polypeptide. By mini-Tn7, cre-loxp transposition and red-gam homologous recombination, BMDS can display up to ten foreign genes together (Yao et al. 2010). In addition, baculovirus exhibits high titer and does not compete with target antigens, which provides an ideal method for the development of polyvaccine. Whether BMDS is capable of supporting the simultaneous expression of even more genes and/or the display of proteins with more complicated biological activities remains to be seen. In light of the results described above, we speculate that BMDS, with its wide range of use, high display efficiency, and multi-gene co-display capabilities, is of potentially important relevance to a variety of biological and genetic applications, whether it is in industry or in the development of novel recombinant vaccine for prevention of epidemics.