The bacmid AcBac-Syn was constructed as described in Materials and Methods. Briefly, plasmid pGF(K)-egfp was first constructed by substituting the CmR and lacZ genes of pGF plasmid with a cassette of KanR, lacZ:attTn7, and egfp genes (Fig. 1A). Then the linearized pGF(K)-egfp and Bsu36I digested AcMNPV-WIV-Syn1 were assembled by TAR in yeast to generate AcBac-Syn (Fig. 1B). The AcBac-Syn is 145.9 kb in size and its complete genome sequence is deposited in GenBank with accession number MW893240.
To demonstrate how to use AcBac-Syn for foreign gene insertion and expression, Dsred was inserted into the AcBac-Syn by transposition as described in Materials and Methods using commercially available vector pFastBac and transposase provided by helper plasmid (Fig. 1C). The resulting bacmid AcBac-Syn-Dsred contained Dsred under polyhedrin promoter and gentamicin resistance gene (GenR) at the attTn7 site (Fig. 1C). The original egfp in AcBac-Syn was still retained in AcBac-Syn-Dsred. The correct insertion of AcBac-Syn-Dsred was confirmed by PCR and sequencing (data not shown).
One of the major differences between AcBac-Syn and the conventional AcMNPV bacmids is that the former induces high copy number of DNA. The pGF(K)-egfp contains two replication origins, ori2 and oriV, responsible for DNA replication in E. coli cells. In the presence of product of trfA gene, genomic DNA replicates from oriV and produce high-copy number; otherwise, genomic DNA replication starts from ori2 at a single-copy number. The E. coli EPI300 strain cells contain an inducible trfA gene, and by adding inducer the DNA copy number of AcBac-Syn could be significantly increased. We compared the genome copy numbers of AcBac-Syn with that of AcBac-egpf-ph which was constructed based on the commercial Bac-to-Bac system (Luckow et al. 1993). As shown in Fig. 2, without inducer CopyControl, the total copy number of AcBac-Syn and AcBac-egfp-ph DNA were 2.57 × 109 and 3.0 × 109, respectively in 3 mL E. coli culture, and there was no significant difference between them (P>0.05); but in the present of CopyControl, the DNA copies of AcBac-Syn (5.99 × 109) was significantly higher than that of AcBacegfp-ph (1.86 × 109) (P < 0.05). The result confirmed that AcBac-Syn is a copy number inducible bacmid in E. coli EPI300.
Figure 2. Comparison of DNA copy numbers of AcBac-Syn and AcBacegfp-ph in E. coli. The E. coli EPI300 containing AcBac-Syn or AcBac-egfp-ph was cultured overnight with or without the presence of CopyControl Induction Solution (CopyControl, Epicentre). Total DNAs of 3 mL each E. coli culture (OD600 value 1.8) were extracted and suspended in 100 μL water. Quantitative PCR was performed to detect the AcBac-egfp-ph or AcBac-Syn DNA copy numbers. The copy numbers were the average value from three independent infections. Error bars represent standard deviations. *P < 0.05, P > 0.05 means no significant difference.
To rescue viruses from bacmids, transfection and infection assays were performed. At 24 hpt, a few of the Sf9 cells transfected with bacmids of AcBac-Syn and AcBac-Syn-Dsred produced green fluorescence; the green fluorescence increased at 48 hpt and could be observed in most cells at 72 hpt (Fig. 3A). The red fluorescence appeared in the AcBac-Syn-Dsred transfected cells at 48 hpt and increased at 72 hpt (Fig. 3A). The supernatants from the transfections of AcBac-Syn and AcBac-Syn-Dsred were collected at 120 hpt and used to infect the Sf9 cells. A mock infection and infections by the viruses of AcMNPV-WT, AcMNPV-WIV-Syn1 and AcMNPV-egfp-ph (control) were performed. At 72 hpi, almost all the cells infected with AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WIV-Syn1 and AcMNPV-egfp-ph showed green fluorescence, and the cells infected with AcBac-Syn-Dsred also produced red fluorescence. The typical cytopathic effect of hypertrophied nuclei and OBs appeared in the cells infected with all the viruses (Fig. 3B). These data proved that AcBac-Syn could produce infectious virus, and the inserted foreign gene (e.g. Dsred) could be expressed in Sf9 cells.
Figure 3. Transfection and infection assays and single-step growth curve analysis. A Transfection of Sf9 insect cells with AcBac-Syn and AcBac-Syn-Dsred. The images were taken at 24, 48 and 72 hpt. Scale bars are presented as 200 μm. B Cytopathic effects of Sf9 cell after virus infection. Sf9 Cells were infected with AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1 and AcBac-egfp-ph at an MOI of 5, or mock infected. Images were taken at 72 hpi. Nuclei were stained with DAPI. Scale bars are presented as 20 μm; C Onestep growth curves. The Sf9 cells were infected with AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1 and AcBacegfp-ph at an MOI of 5; the supernatants were collected at 0, 12, 24, 48, 72 and 96 hpi, and titrated by EPDA. The titer, transformed logarithmically, was the average titer from three independent infections. Error bars represent standard deviations.
To investigate the proliferation property of AcBac-Syn in Sf9 cells, one step growth curves of AcBac-Syn, AcBacSyn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1, and AcBac-egfp-ph were detected. All the viruses had identical kinetics of infection in which a logarithmic phase lasted up to 48 hpi and a stationary phase continued from 48 to 96 hpi (Fig. 3C). Statistical analysis results indicated that there was no significant difference between the titers of the five viruses at each time point (P > 0.05).
To compare the morphology of OBs among AcBac-Syn, AcBac-egfp-ph and AcMNPV-WT, the OBs of all the viruses were purified from the infected S. exigua larvae and observed by scanning electron microscope (SEM) and transmission electron microscopy (TEM). SEM results showed that the OBs of both viruses had smooth surface and polyhedral shape, and the sizes of OBs from different viruses were similar (Fig. 4). TEM results showed that like control viruses, OBs of AcBac-Syn contained multiple matured (enveloped) ODVs (Fig. 4). So morphologically the OBs of AcBac-Syn were similar to that of the control viruses.
Figure 4. Electron micrographs of OBs of AcBac-Syn, AcMNPVWT and AcBac-egfp-ph. After one round of replication in S. exigua larvae, the OBs of AcBac-Syn, AcMNPV-WT and AcBac-egfp-ph were purified and processed for SEM and TEM observation. Scale bars are presented as 2 μm (SEM) and 500 nm (TEM), respectively.
The oral infection assay of AcBac-Syn and AcMNPVWT was performed in third instar S. exigua larvae. The LC50 for AcBac-Syn and AcMNPV-WT were 8.90 × 106 OBs/mL and 1.69 × 107 OBs/mL, respectively (Table 1). Potency ratio value, performed by dividing the LC50 of the AcBac-Syn by that of AcMNPV-WT, was 1.78 with the 95% confidence interval (95% CI) from 0.93 to 3.66. The 95% CI of potency ratio included 1.0, which indicated that there was no significant biological difference between the two viruses.
Virus LD50(95% CI)
Slope(95% CI) χ2/d.f Potency ratioa(95% CI) AcMNPV-WT 16.91(9.85–39.86) 0.85(0.58–1.13) 0.64 1.78(0.93–3.66) AcBac-Syn 8.90(5.66–16.20) 0.91(0.65–1.18) 0.39 aPotency ratio was calculated by dividing the LC50 value of the AcBac-Syn by that of AcMNPV-WT.
Table 1. Oral infection analysis of AcMNPV-WT and AcBacSyn.
To evaluate OBs production of AcBac-Syn, Sf9 cells were infected by the BV of AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1, and AcBac-egfp-ph at the dose of 5 MOI, respectively. The formation of OBs was observed at 72 hpi, and the results showed that almost all cells produced OBs in nuclei when infected by AcBacSyn, AcBac-Syn-Dsred, AcMNPV-WT and AcMNPVWIV-Syn1, however, only a few AcBac-egfp-ph infected cells formed OBs (Fig. 5A). At 96 hpi, the infected cells were harvested and the polyhedrin protein (PH) was detected by Western blot. In agreement with the above observation, the yields of PH in the AcBac-Syn, AcBacSyn-Dsred, AcMNPV-WT and AcMNPV-WIV-Syn1 infected cells were comparable and significantly higher than that in AcBac-egfp-ph infected cells (Fig. 5B). Therefore, in terms of the yields of OB and PH, the AcBacSyn virus was comparable to AcMNPV-WT, and better than AcBac-egfp-ph.
Figure 5. Polyhedrin production in AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WIV-Syn1, AcMNPV-WT and AcBac-egfp-ph infected Sf9 cells. A Image of cells at 96 hpi. 3 × 106 Sf9 cells were infected with BVs of AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1 and AcBac-egfp-ph at an MOI of 5, or mock infected. Scale bars are presented as 20 μm. B Detection of polyhedrin protein by Western blot analysis. 3 × 106 Sf9 cells were infected with BVs of AcBac-Syn, AcBac-Syn-Dsred, AcMNPV-WT, AcMNPV-WIV-Syn1 and AcBac-egfp-ph at an MOI of 5, or mock uninfected. The cells were harvested at 72 hpi and Western blot assay was performed. Antibodies against AcMNPV Polyhedrin (anti-PH), ODV-E25 (anti-E25) or anti-GAPDH were used as primary antibodies, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma) was used as secondary antibody.