Codon optimization, expression in Escherichia coli, and immunogenicity analysis of deformed wing virus (DWV) structural protein

Abstract

Background: Deformed wing virus (DWV) is a serious threat to honey bees (Apis mellifera) and is considered a major cause of elevated losses of honey bee colonies. However, lack of information on the immunogenicity of DWV structural proteins has hindered the development of effective biocontrol drugs.

Methods: We optimized the VP1, VP2 and VP3 codons of DWV surface capsid protein genes on the basis of an Escherichia coli codon bias, and the optimized genes of roVP1, roVP2 and roVP3 were separately expressed in E. coli and purified. Next, the three recombinant proteins of roVP1, roVP2 and roVP3 were intramuscularly injected into BALB/c and the immunogenicity was evaluated by the levels of specific IgG and cytokines. Furthermore, anti-roVP-antisera (roVP1 or roVP2 or roVP3) from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test, respectively.

Results: The optimized genes roVP1, roVP2 and roVP3 achieved the expression in E. coli using SDS-PAGE and Western blotting. Post-immunization, roVP2 and roVP3 exhibited higher immunogenicity than roVP1 and stimulated a stronger humoral immune response in the mice, which showed that the recombinant proteins of roVP3 and roVP2 induced a specific immune response in the mice. In the challenge test, data regarding quantitative real-time RT-PCR (qRT-PCR) from challenged pupae showed that the level of virus copies in the recombinant protein groups was significantly lower than that of the virus-only group at 96 h post-inoculation (P < 0.05). Among them, the degree of neutralization using antibodies raised to the recombinant proteins are between approximately 2-fold and 4-fold and the virus copies of the roVP3 group are the lowest in the three recombinant protein groups, which indicated that specific antibodies against recombinant proteins roVP1, roVP2 and roVP3 of DWV could neutralize DWV to reduce the virus titer in the pupae. Collectively, these results demonstrated that the surface capsid protein of DWV acted as candidates for the development of therapeutic antibodies against the virus.

Keywords: Challenge test; Codon optimization; DWV; Immune responses; Structural proteins.

Figure 1. Expression and purification of the recombinant proteins by SDS-PAGE analysis in E. coli.

(A)–(C) represent the recombinant proteins roVP1, roVP2 and roVP3, respectively. Lane 1; protein weight marker. Lane 2–4; induced E. coli BL21 (DE3) of the corresponding plasmids after 6 h with 0.1 mM, 0.25 mM and 0.5 mM IPTG, respectively. Lane 5; uninduced E. coli BL21 (DE3) of the corresponding plasmids as control. Lane 6; purification of the corresponding recombinant proteins.

Figure 2. Western blot identification of the recombinant proteins with the anti-His tag antibody and mouse anti-DWV polyclonal antibody.

(A)–(C) represent roVP1, roVP2 and roVP3, respectively. Lane 1; anti-His tag antibody was used as primary antibody. Lane 2; mouse anti-DWV polyclonal antibodies as primary antibody. Lane 3; negative control (NC), the serum of healthy mice.

Figure 3. Specific IgG antibodies in serum of the immunized mice.

The OD₄₅₀ value of total IgG were detected on 0, 2, 4 and 6 weeks post-vaccination. Data are expressed as the mean ± SD (n = 5) values. *Significantly different compared with the control group (P < 0.05). **Significant differences between three groups immunized with recombinant proteins (P < 0.05).

Figure 4. Lymphocyte proliferation responses of immunized mice.

The values of the stimulation index (SI) were calculated at 6 weeks post-vaccination. Data are expressed as the mean ± SD (n = 5) values. *Significantly different than the control group (P < 0.05).

Figure 5. Cytokine response in sera of immunized mice.

(A)–(D) represent the levels of cytokines IL-2, IL-4, IL-10 and IFN-γ by ELISA, respectively. Data are expressed as the mean ± SD (n = 5) values. *Significantly different compared with the control group (P < 0.05). **Significant differences between three groups immunized with recombinant proteins (P < 0.05).

Figure 6. Relative normalized DWV of the honeybee pupae.

The quantification of DWV titer in the samples was performed by the real time qRT-PCR, and the relative normalized DWV titer was determined as the DWV titer after normalization with the reference gene Actin and the relative DWV titers of the pupae were assessed by calculating delta Ct values (ΔCt = Ct_(DWV) − Ct_(actin)). *Significantly different compared with the virus-only group (P < 0.05). **Significant differences between three groups immunized with recombinant proteins (P < 0.05).