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In order to express the VP2 protein in vitro, the fulllength open reading frame (ORF) of VP2, with the addition of a His-tag sequence designed and incorporated into the N-terminus, was inserted into a pFastBac-1 vector, downstream of the polyhedrin (ph) promoter (as shown schematically in Figure 1A). The recombinant pFbGCRV-VP2 plasmid was purified and used to transform DH10Bac competent cells for transposition. The recombinant bacmid AcGCRV-His-VP2 was identified by PCR, using either M13 primers or gene-specific primers. As shown in Figure 1B, three specific bands with molecular weights of around 6.0 kb were identified in the recombinant bacmid by PCR amplification. Only a 0.3kb band was amplified in the empty bacmid. This result suggested that the VP2 gene had been transposed successfully into the recombinant bacmid.
Figure 1. Construction and identification of recombinant AcGCRV-His-VP2 bacmid. A: Schematic representation of the construction of the AcGCRV-His-VP2 bacmid. B: Identification of recombinant AcGCRV-His-VP2. M, 1kb DNA gene ruler; 1, positive amplification with M13-S+M13-AS (~2.3+3.8kb); 2, empty bacmid amplification with M13-S+ M13-AS (~0.3kb); 3 and 4, positive amplification using M13-S+GCRV/S2-AS (~1.7+3.8kb) and GCRV/S2-S+M13-AS (~3.8+0.6kb) primer pairs, respectively; 5, ddH2O control, amplification with M13-S+M13-AS.
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Sf9 cells were transfected with the recombinant bacmid AcGCRV-His-VP2 to produce recombinant baculovirus passage 1 (P1). P1 stock was clarified by centrifugation and used to infect Sf9 cells at an MOI of 2.5 to generate a high-titer baculovirus stock (P2-P5). Visualization of the intracellular distribution of VP2 was performed by immune-staining with His-tag monoclonal and anti-VP2 polyclonal antibodies, using fluorescence microscopy. Analysis of infected cells revealed that His-VP2 could be detected at 24 h p.i. As shown in Figure 2A, at 24 h p.i. the expression of His-VP2 appeared as small spots of immunofluorescence that were scattered within the infected cells. As infection progressed, at 48 h and 72 h, many large immunofluorescent spots were observed. At 72-96 h p.i., infected cell suspension cultures were collected for the identification of secreted VP2, by IB analysis using anti-VP2 polyclonal antibody. The IB results showed that the specific target band of about 140 kDa, corresponding to the molecular weight of the VP2 protein, was detected in both recombinant baculovirus-infected Sf9 cell lysates and in pelleted supernatant samples (Figure 2B). No such band was detected in mock-infected cells. The IF and IB results therefore indicated that the recombinant protein of interest was correctly expressed in Sf9 cells and that, following expression, the recombinant VP2 protein was secreted into the medium.
Figure 2. Identification of His-VP2 protein expression in Sf9 cells. A: Mock-infected Sf9 monolayers and Sf9 monolayers infected with a recombinant baculovirus were fixed at 24, 48 and 72 h p.i. and co-immunostained with His-tag monoclonal and VP2 polyclonal antibodies, followed by immunostaining with Alexa 568 anti-mouse IgG (Red) and Alexa 488 anti-rabbit IgG (Green); the nuclei were then counterstained with Hoechst stain. B: Immunoblotting analysis of VP2 expression, using antibody to VP2. 1 and 2, mock-infected and infected Sf9 cell lysate, respectively; 3 and 4, mock-infected and infected Sf9 culture supernatant pellet, respectively; 5 and 6, infected and mock-infected CIK cell lysate.
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To investigate the function of GCRV VP2 in the absence of other viral or cellular proteins, the soluble His-tagged VP2 was purified by using a Ni2+-chelating resin column. We also prepared purified GCRV particles and rVP2390-900 as controls. As shown in Figure 3, rVP2 was purified and compared both to GCRV particle components and to rVP2390-900. Protein bands of 140 kDa were visualized by SDS-PAGE and Coomassieblue staining for both the purified GCRV and AcGCRVHis-VP2 samples, in contrast to the 55 kDa protein band observed for the rVP2390-900 positive control (Figure 3A). IB assays using VP2-specific polyclonal antiserum further confirmed these SDS-PAGE results (Figure 3B). In addition, the image from the highly purified GCRV particle demonstrated that cell components were absent from the virion sample (Figure 3C). Consequently, all the purified VP2-related samples were suitable for further replicase assays.
Figure 3. Purification of His-tagged recombinant proteins and virus particles. A: Purified virus particles, rVP2 and rVP2390-900 (shown in lanes 1-3) were subjected to SDS-PAGE, in each case loading 10 μL of sample onto the gel. B: Immunoblot analysis of virus particles and purified rVP2 protein, using antibody to VP2; lanes 1'-3' correspond to lanes 1-3 in A. C: Negatively stained electron micrographs of purified virus particle.
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As a first step towards establishing an assay system for rVP2, we initially used the poly (C)-dependent, oligo (G)-primed poly (G) polymerase assay (Starnes M C, et al., 1993). In the standard polymerase assay, the enzymatic activity of rVP2 is quantified as the rate of incorporation of [a-32P]-GTP into precipitable material in response to a poly (C) template and an oligo(G) primer. Besides assaying rVP2, we also assayed rVP2390-900 and purified GCRV particles. As shown in Table 1, using poly (C)/ oligo (G) 12-18 as template, there was incorporation of [a-32P]-GTP into a newly synthesized RNA with either rVP2 or GCRV. However, this incorporation was decreased by adding VP2 antiserum to the rVP2 or GCRV reactions (Table 1), indicating that the catalytic activity of the RNA polymerase was inhibited by specific VP2 antiserum. In addition, the incorporation of [a-32P]-GTP was dependent upon the presence of poly (C), because no [a-32P]-GTP incorporation was detected in the absence of poly (C) template (data not shown). No significant incorporation was detected with the purified rVP2390-900.
Sample
(100ng/each)Template/primer 32P-GTP incorporation
(cpm 10-3)Virion Poly(C)/Oligo(G)
Poly(C)/Oligo(G)+VP2 serum21.54
11.70rVP2 Poly(C)/Oligo(G)
Poly(C)/Oligo(G)+VP2 serum16.06
4.35rVP2390-900 Poly(C)/Oligo(G) 0.74 Table 1. Poly (G) polymerase activity assays
The replicase activity of rVP2 was characterized further by investigating the effects of temperature and of the concentration of the divalent cation, Mg2+. The optimal reaction temperature for the incorporation of [a-32P]-GTP into the poly (C) template was 28 ℃ (Figure 4A). Polymerase activity increased gradually as the Mg2+ concentration was raised from 1mmol/L to 10 mmol/L, but was largely inhibited when the concentration was increased further to 50 mmol/L (Figure 4B).
Figure 4. Characterization of RNA polymerase of rVP2. RdRp reactions with 100 ng of purified rVP2 were carried out as detailed in Materials and Methods. Radioactivity was measured by scintillation counting. A: Effect of temperature on replicase activity. B: Effect of Mg2+ concentration on replicase activity.