| Rabbit hemorrhagic disease (RHD) is a disease of domestic and wild rabbits, with relevant economic and ecologic importance caused by Rabbit hemorrhagic disease virus (RHDV). The prototype strain of Lagovirus,RHDV, belongs to the Caliciviridae family. Mature RHDV virions are spherical, non-enveloped particles with a T=3, icosahedral capsid whose outer diameter varies between 32 and 44 nm. The capsid protein (VP60) expressed in insect cells could self-assemble into virus like particles (VLPs). RHDV-VLPs have successfully been shown to be good platforms for carrying foreign epitopes.The aim of present study was to investigate the potential of VLPs from RHDV as a delivery system for foreign epitopes. Three chimeric RHDV-VLPs incorporating double CD8+ T cell epitopes derived from chicken ovalbumin (OVA) were generated. To evaluate the immune responses induced by the chimeric proteins, the titers of anti-VP60 specific antibody were detected by an indirect ELISA. In addition, spleen cells of immunized mice were used to detect the specific IFN-y production by ELISPOT.1. Construction of three shuttle vectorsThree chimeric RHDV-VLPs incorporating double CD8+T cell epitopes derived from chicken ovalbumin (OVA) were generated. Two of the integrations were performed by replacing aa.2-13(DN2) and 302-309 (DC) of VP60 with OVA epitopes respectively, and another one was performed by inserting the epitopes into N-terminus of the VP60 (DN1). Three recombinant shuttle vectors were respectively constructed successfully, named as bamcid-DN1, bamcid-DN2 and bamcid-DC.2. Expression and identification of the three chimeric proteinsThree recombinant shuttle vectors (bacmid-DN1, bacmid-DN2 and bacmid-DC) were transfected into Sf9 cells with lipofectamine. Three recombinant baculoviruses (rBac-DN1, rBac-DN2, rBac-DC) were harvested when the siginifican cytopathic effect were found. These chimeric proteins (DN1, DN2 and DC) were expressed successfully in baculovirus expression system and analyzed by IFA, SDS-PAGE and Western blot. The results indicated that all the chimeric proteins were effectively expressed and had strong VP60-specific antigenicity.3. Electron microscopic and immunogenicity of the chimeric proteinsThe three chimeric proteins (DN1, DN2 and DC) were observed by electron microscopic after being primary purificated. The C57BL/6 mice were immunized with chimeric proteins suspended in PBS without adjuvant. To evaluate the immune responses induced by the chimeric proteins, the titers of anti-VP60 specific antibody were detected by an indirect ELISA. In addition, spleen cells of immunized mice were used to detect the specific IFN-γ production by ELISPOT. Interestingly, no significant difference in anti-VP60 specific antibody titers were found between the VP60 group and chimeric protein groups (p> 0.05), While different chimeric proteins could induce varied levels of specific IFN-γ production.Three chimeric RHDV-VLPs incorporating double CD8+T cell epitopes (GSSIINFEKLGSSIINFEKLGS) derived from chicken ovalbumin (OVA) were generated. Integrations were performed at the following locations:1) inserting at the N-terminus of the capsid protein VP60 (DN1); 2) replacing amino acid positions 2-13 of VP60 (DN2); 3) replacing amino acid positions 302-309 of the VP60 (DC). Three chimeric proteins were correctly expressed in baculovirus system and could correctly self-assemble into VLPs. All of the chimeric proteins could induce the high levels of anti-VP60 specific antibody titers and specific IFN-y production in the absence of any adjuvant in the murine model. The anti-VP60 specific antibody titers of chimeric protein groups were comparable with that of the group VP60 (P>0.05). Mice injected with DN1, DN2 and DC showed a significant higher production of IFN-y-secreting cells than that in group VP60 (p<0.01). This manuscript should be very valuable for enhancing the understanding of using RHDV-VLPs as a carrier for foreign genes. Meanwhile, the length of the foreign genes which RHDV-VLPs carried was extended. |
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