Cells of Helicoverpa zea cell line Hz-AM1 were maintained in Grace medium (Invitrogen, USA), supplemented with 10 % fetal bovine serum. The HearNPV vHaBac-egfp-ph (Song J J et al., 2008), was used and was propagated in Hz-AM1 cells. The vector pMAL-C2x (New England Biolabs, USA) and the E.coli strain TB1 were used for the expression of recombinant proteins, with the exception of truncated P74 (residues 2-219), which was expressed using pET-32a and E.coli strain BL21.
Based on previous investigations of the proteolytic cleavage of AcMNPV P74 (Peng K et al., 2011; Slack J M et al., 2008), a truncated HearNPV P74 (2-460 aa) lacking all three TM domains, P74△3TM, was constructed and further dissected at the proposed cleavage sites into three non-overlapping segments, designated N, M and C (Fig. 1). Segment N was then dissected into peptide fragments Ⅰ, Ⅱ, Ⅲ and Ⅳ, such that there was an overlap of 25 amino acids between each pair of adjacent peptides (Fig. 1). Fragments Ⅲ and Ⅳ were further divided into three and four fragments, respectively, such that between each pair of adjacent peptides, there was an overlap of eight amino acids. Each truncated peptide was fused with fragment Ⅰ at its C terminus. The resulting chimeric P74 derivatives were designated Ⅲ F1-F3 and Ⅳ F1-F4 (Fig. 1). Finally, Ⅲ F3 was dissected into Ⅲ F3 (1)-(5) and Ⅳ F4 was dissected into Ⅳ F4 (1)-(7), which in each case were fused with fragment Ⅰ at the C terminus (Fig. 1).
Figure 1. Schematic diagram of HearNPV P74 fragments. The full-length structure of HearNPV P74 is shown at the top. The two putative cleavage regions of P74 are indicated by arrowheads. The three conserved C-terminal transmembrane domains predicted by the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/) are shown in red and indicated as TM. The names of the truncated and chimeric P74 fragments are indicated within, or to the left of, the corresponding rectangles. The location of each fragment within HearNPV P74 is indicated beneath.
The gene fragments encoding the above peptides were PCR-amplified using the primer pairs listed in Table 1. All PCR-amplified DNAs, except for that encoding N, were cloned into pMAL-C2x between BamH Ⅰ and Hind Ⅲ sites and expressed in E.coli strain TB1. DNA encoding the N fragment was cloned instead into pET-32a and expressed in E.coli strain BL21. Proteins were expressed and purified according to a standard protocol (NEB).
Fragementsa Primersb, c, d P74△3TM F CGCGAATTCTTCATGTCGAATATCATTTATATAC R GCGAAGCTTTTATTTAATTGCTATTTTAGTCATC N F GCCGGATTCTTCATGTCGAATACATTTATATACA R CGGCTCGGGCAATAGGCTTCGTTGAAAAAAA M F CGCGGATCCCTAGGCGATACTGTACTTGTC R CCCAAGCTTGCGCGTTGCCATCTATCCGTTG C F CGCGGATCCCGTTCCTTACAACCGAACGG R CCCAAGCTTGCCGCCGTAACGGTTTTAATAGC Ⅰ F CGCGGATCCTTCATGTCGAATATCATTTATATAC R CCCAAGCTTGCGTAAAAATCATCATTGT TAGCCG Ⅱ F CGCGGATCCTTCATACCGAAATGGCGGG R CCCAAGCTTGCCGATGTTTGTGTATACCCGAA Ⅲ F CGCGGATCCGAAAGCATGAGTTGTTATCCG R CCCAAGCTTGCTGTCAAAGTGTCCACTATAATAC Ⅳ F CGCGGATCCGGTGCCGAAAATGAAATACAAA R CCCAAGCTTGCGCAATAGGCTTCGTTGAAAAAA Ⅲ F1 F CGCGGATCCCAGTTCGGGTATACACAAACATCGGAAACGGCAGTGGCGTACGCTCAACCCGCATTCATGTCGAATATCATTTATATAC Ⅲ F2 F CGCGGATCCGCAGTGGCGTACGCTCAACCCGCATGCTACAATTTGGACAGGGCGGCGGCGGTGTTCATGTCGAATATCATTTATATAC Ⅲ F3 F CGCGGATCCAATTTGGACAGGGCGGCGGCGGTGCGCGACGGTGCCGAAAATGAAATACAAACGTTCATGTCGAATATCATTTATATAC Ⅳ F1 F CGCGGATCCAAATGTATTATAGTGGACACTTTGACAAAAATGTATTTGAACACTCCCTATTTGCGTACCCAACCTACTGTACCTACGCC Ⅳ F2 F CGCGGATCCTTGAACACTCCCTATTTGCGTACCGATGACCATTTGATACAGGGCGTTGATGATGTGCCCCAACCTACTGTACCTACGCC Ⅳ F3 F CGCGGATCCATACAGGGCGTTGATGATGTGCCCGGATTCAATGTGACAAACGATACGGATCAACTTTTTCAACCTACTGTACCTACGCC Ⅳ F4 F CGCGGATCCACAAACGATACGGATCAACTTTTTCCCGAAAGATTCGAAGGTTTTTTCAACGAAGCCTATTGCCAACCTACTGTACCTACGCC Ⅲ F3(1) F CGCGGATCCAATTTGGACAGGGCGGCGGCGGTGCGCTTCATGTCGAATATCATTTATATAC Ⅲ F3(2) F CGCGGATCCGACAGGGCGGCGGCGGTGCGCGACGGTTCATGTCGAATATCATTTATATAC Ⅲ F3(3) F CGCGGATCCGCGGCGGCGGTGCGCGACGGTGCCGAATTCATGTCGAATATCATTTATATAC Ⅲ F3(4) F CGCGGATCCGCGGTGCGCGACGGTGCCGAAAATGAATTCATGTCGAATATCATTTATATAC Ⅲ F3(5) F CGCGGATCCCGCGACGGTGCCGAAAATGAAATACAAACGTTCATGTCGAATATCATTTATATAC Ⅳ F4(1) F CGCGGATCCACAAACGATACGGATCAACTTTTTCCCTTCATGTCGAATATCATTTATATAC Ⅳ F4(2) F CGCGGATCCGATACGGATCAACTTTTTCCCGAAAGATTCATGTCGAATATCATTTATATAC Ⅳ F4(3) F CGCGGATCCGATCAACTTTTTCCCGAAAGATTCGAATTCATGTCGAATATCATTTATATAC Ⅳ F4(4) F CGCGGATCCCTTTTTCCCGAAAGATTCGAAGGTTTTTTCATGTCGAATATCATTTATATAC Ⅳ F4(5) F CGCGGATCCCCCGAAAGATTCGAAGGTTTTTTCAACTTCATGTCGAATATCATTTATATAC Ⅳ F4(6) F CGCGGATCCAGATTCGAAGGTTTTTTCAACGAAGCCTTCATGTCGAATATCATTTATATAC Ⅳ F4(7) F CGCGGATCCGAAGGTTTTTTCAACGAAGCTATTGCTTCATGTCGAATATCATTTATATAC a The reverse primer used for the amplification of fragments Ⅲ F1 - Ⅳ F4(7) was the same as that used for the amplification of fragment Ⅰ; b Primer sequences are 5′ to 3′; c Restriction enzyme sites are underlined; d F indicates forward primer; R indicates reverse primer.
Table 1. Primers for the PCR amplification of truncated and chimeric P74 gene fragments
Monoclonal antibodies against HearNPV P74 were prepared as reported in the literature (Evan G I et al., 1985). BALB/c mice were immunized with purified HearNPV P74△3TM. Spleen cells were then harvested from the immunized mice and fused with SP2/0 myeloma cells. Cell culture supernatants were screened by Western blotting, using HearNPV ODV (data not shown). Positive hybridomas were selected and further cloned and then inoculated into syngeneic mice, following the protocol approved by the Animal Ethics Committee at the Wuhan Institute of Virology, Chinese Academy of Sciences (CAS). Ascites fluids from three clones were withdrawn and clarified, and designated as 20D9, 20F9 and 21E1.
The mAbs were purified from collected ascites fluids using a Protein A-SepharoseTM column. Ascites fluids were each diluted with an equal volume of phosphate-buffered saline (PBS), pH 8.0; each was then applied to the column and the column was then in each case washed with 10 column volumes of PBS (pH 8.0); the antibodies were then eluted with 100 mmol/L citric acid buffer (8.61 g citrate and 2.64 g citric acid sodium in 500 mL H2O, pH 3.0). Each eluate was mixed with 2/5 elution volume of 1 mol/L Tris/HCl buffer, pH 8.0. The concentrations of the resulting purified mAb preparations were adjusted to 1.5 mg/mL.
Purified prokaryotic expressed proteins were separated by 12% SDS-PAGE, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Germany). The anti-P74 polyclonal antibody (Song J J et al., 2008) from the supernatant solution of P74 hybridoma or ascites fluids was used as the primary antibody and alkaline phosphatase (AP)-conjugated anti-mouse immunoglobulin G(Protein Tec Group, USA) was used as the secondary antibody. Signals were detected as previously described (Deng F et al., 2007).
Hz-AM1 cells (3×105) were infected with vHaBacHZ8-egfp-ph at a multiplicity of infection (MOI) of 10. At 48 h postinfection (p.i.), the HzAM1 cells, on 10 mm glass plates, were washed three times with PBS, fixed with 4 % (w/v) paraformaldehyde and then permeabilized with fresh 0.15 %(w/v)Triton X-100 (in PBS). The cells were then incubated with primary antibodies (anti-P74 polyclonal antibody, supernatant solution from P74 hybridoma or ascites fluids, or purified mAbs) at 4 ℃ overnight. DyLightTM 594 or Rhodamine-conjugated anti-mouse IgG (Jackson ImmunoResearch, USA) was then added and the cells were incubated at 25 ℃ for 2 h, before being washed three times with PBS. Hoechst 33258 (Sigma, USA) was used to stain the nucleus for 15 min at 25 ℃. After washing with PBS, the cells were examined using a Revolution XD confocal microscope (Andor Technology, UK).