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Abstract Cydia pomonella granulovirus(CpGV) has been developed as one of the most efficient, environmental and safe insecticides for control of the codling moth. To enrich the research contents of CpGV GP37 protein and explore mechanism of synergism of Nucleopolyheroviruses(NPVs) and Bacillus thuringiensis (Bt) by the GP37 protein, a series of recombinant Autographa californica multiple nucleocapsid NPV (AcMNPV) bacmids containing egfp or CpGV gp37 fused with egfp were constructed. The resulting bacmids were named vAcGP37, vAcGP37EGFP, vAcEGFPGP37, and vAcEGFP, respectively. In vAcGP37EGFP and vAcEGFPGP37, EGFP was fused in frame with C terminal and N terminal, respectively. The bacmid vAcGP37 expressed GP37 protein and vAcEGFP expressed EGFP only. All recombinant bacmids were confirmed by PCR. Sf9 cells were transfected with the recombinant bacmids, and supernatants containing budded virus were collected at 96 h after transfection. Examinations were carried out for protein expression by Western blotting analysis using anti-GFP and anti-GP37 as primary antibody following SDS-PAGE of the cell lysates. An fusion protein about 50 kD, which was in consistence with the size of GP37 and EGFP, was detected in the cell lysates transfected with vAcGP37EGFP or vAcEGFPGP37. By contrast, an approximate 25 and 30 kD protein were detected in those cells lysates transfected with vAcGP37 or vAcEGFP, respectively. Sf9 cells (1×105) in 35-mm glass bottom cell culture dishes were infected with vAcGP37, vAcGP37EGFP, vAcEGFPGP37, and vAcEGFP, respectively. At 24 h after transfection, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed with PBS and then dealled with 0.25% Triton X-100 for 3 min at room temperature. Following a wash with PBS, the cells were stained with Hoechst 33258 working reagent for 5 min at room temperature. Cells were washed three times with PBS and visualized with confocal laser scanning microscope for fluorescence, respectively. In cells infected with vAcEGFP, EGFP was expressed without fusion of GP37 and fluorescence was dispersed uniformly throughout the cytoplasm and nucleus. In contrast, in cells infected with vAcGP37EGFP and vAcEGFPGP37, EGFP was expressed with fusion of GP37 and fluorescence distributed mainly in the cytoplasm. These results suggested that CpGV GP37 protein was localized mainly in the cytoplasm. The eukaryotic expression of CpGV GP37 protein may provide conditions for further study of this protein. The subcellular localization of CpGV GP37 protein in infected cells can lay a foundation for the study of synergic mechanism of this protein and enrich the theory of baculovirus GP37 protein.
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Received: 31 December 2013
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