Expression And Immunogenicity Of Rabbit Hemorrhagic Disease Virus-Like Particles Carrying Cd8~+T Cell Epitopes Form OVA

Rabbit hemorrhagic disease (RHD) is a highly contagious and fatal disease caused by Rabbit hemorrhagic disease virus (RHDV). RHDV, the etiological agent of RHD, is a member of the family Caliciviridae. Despite the fact that it has a simple structure, only one major structural protein, many studies revealed that RHDV can induce strong humoral, cellular and mucosal immune responses. In addition, it has a very strong ability to induce interferon. In order to explore the capacity of accommodating insertion of foreign amino acid sequences to RHDV-VLPs, we performed various deletions in different parts of the structural protein VP60, and to which OVA257-264CD8+T cell epitopes (SIINFEKL) were inserted. A series of RHDV-VLPs were generated and analysed by means of transmission electron microscopy, immunology and bioinformatics methods for studying the formation of VLPs and the length, conformation and antigenicity of the foreign epitopes. This study will evaluate the possibility of using RHDV-VLPs as foreign gene carriers and also provide useful reference for researching the formation mechanism of VP60and other related studies of VLPs.The main experiments and results are as follows:1. Construction of shuttle vectorsRecombinant sequences obtained using genesplicing by overlap extension (gene SOEing) and PCR. The target genes were encloned into pMD19-T Vector. The ligated products were transformed into the cell of Escherichia coli, which were filtrated with Ampenicillin on Luria-Bertani medium. After being confirmed by enzyme digestion, the target genes were inserted into eukaryotic vector. The recombinant donor plasmids were transformed into Escherichia coli DH10Bac and the target genes was integrated into Bacmid by screening with blue-white method and determined by PCR. In our research, five recombinant shuttle vectors were constructed successfully, named as N1, N2, I1,12and D1, respectively. 2. Expression and analysis of the five chimeric proteinsThe recombinant shuttle vectors were transfected into monolayer Sf9cells with lipofectamine2000. The siginifican cytopathic effect (CPE) was found in Sf9cell culture infected with recombinant shuttle vectors after3～5days incubation. Recombiant baculovirus were harvested. The RNA was extracted with RNAout Kit method and the target genes were determined by RT-PCR. The positive virus stocked and expressed. The expressed chimeric proteins were assayed by IFA, HA, SDS-PAGE and Western-blot. The generations of the five recombinant baculovirus were assayed by RT-PCR; SDS-PAGE analysis showed that the expressed proteins were about60kDa in size. Immunogenicity of the five recombinant proteins was identified by immunofluorescence, Western blotting, with monoclonal antibody of RHDV (A3C). In hemagglutination assays, only N1and N2were able to cause agglutination of human’O’erythrocytes.3. Space conformation and immunogenicity of the chimeric proteins The five chimeric proteins were successfully expressed in Bac-to-Bac baculovirus expression system. After being primary purificated, the five chimeric proteins(N1、 N2、I1、12and D1) were observated by Electron microscopic. The C57BL/6mice were immunized with chimeric proteins(N1、N2、I1and12), suspended in PBS without adjuvant. RHDV-VLPs-VP60in PBS without adjuvant and sterile PBS were used as controls. The titers of antibody against VP60were measured by indirect ELISA at Od,28d and42d. Two weeks after the second and the third inoculation with RHDV-VLPs-OVA, specific IFN-y-secreting cells were detected in spleens of mice by ELISPOT. The results showed that the chimeric proteins Nland N2can correctly assembled into virus-like particles (VLPs) which was morphologically and structurelly similar to native RHDV-VLPs. VLPs of I1and12are smaller. The virus-like particles of D1are few in number. Chimeric proteins I1and12could induce stronger anti-VP60humoral immunes and more specific IFN-y-secreting cells thanN1andN2.Here we report the generation of recombinant chimeric RHDV-VLPs incorporating a well defined CD8+T cell epitope corresponding to aa257-264(SIINFEKL) from chicken ovalbumin (OVA). This epitope is restricted for MHC classl H-2Kb presentation. The foreign epitope was set at four different locations:1) inserted at the N-terminus of VP60protein(N1);2) replaced amino acid positions2and14of VP60protein (N2);3) deleted and replaced amino acid positions196and207of VP60protein (D1and I1); and4) replaced amino acid positions217and228of VP60protein(I2). Four chimeric RHDV-VLPs were also analyzed as vaccine vectors in the total absence of adjuvant. Interestingly, RHDV-VLPs-OVA were able to stimulate specific IFNy-producing cell priming and to generate powerful and specific anti-VP60humoral immunes. All above results had laid an important theoretical foundation for the possibility of RHDV VLPs as multivalent vaccines vector, and these results will be helpful to research the RHDV VLPs display system.