Construction And Immunogenicity Of Chimeric Proteins Constructed By Ova T Cell Epitope Inserted Into The Capsid Protein VP60of Rabbit Hemorrhagic Disease Virus

Rabbit Hemorrhagic Disease (RHD), caused by Rabbit haemorrhagic disease virus, is characterized by acute, highly contagious, systemic solid organ bleeding and a large area of death in rabbits. RHDV, a member of the calicivirus, could induce strong humoral, cellular and mucosal immune responses in rabbits. VP60is a major viral structural protein, which could be related to anti-viral immune response during RHDV infection. The capsid protein of RHDV spontaneously assembles into VLP which are morphologically and antigenically indistinguishable from native virions, but devoid of the viral RNA. Previous researches have already demonstrated that nonadjuvanted VLPs could induce robust humoral and cell-mediated immune responses. More recent research has also indicated that RHDV might be used as an immune stimulator and self-adjuvant. Researches have shown that VLPs could be excellent candidates to induce a potent immune response to foreign antigens inserted in their particulate structure and to be a vaccine vector.In order to explore the capacity of rabbit hemorrhagic disease virus-like particles (RHDV-VLPs) accommodating exogenous antigen epitopes, the T-cell epitope (fragment of ovalbumin, OVA) was embedded into RHDV VLPs. The chimeric proteins generated were tested for assembly into VLPs, for antigenicity and immunogenicity, including their capacity to induce immune responses, and for their capacity to present the foreign epitope.Major tests and results are descirbed as follow:1. Construction and identification of the recombinant shuttle vectorsThe CD8+T cell epitope (SIINFEKL) were inserted into the different sites of RHDV VLPs:1) directly inserted into the VP60C-terminal;2) replaced the VP60502-510amino acid of VP60;3) replaced the VP60411-417amino acid of VP60;4) replaced the VP60302-309amino acid of VP60. The sequences coding chimeric proteins were obtained by PCR or SOE PCR. The PCR product was digested and cloned into corresponding enzyme digested pFastBacTM HT1plasmid. The four recombinant transfer vectors were transformed into E.coli DH10Bac. The colonies of E.coli containing recombinant Bacmids were screened three times by blue/white selection. All the recombinant Bacmids were characterized by PCR using the M13forward and reverse primers, and verified by sequencing. The recombinant plasmids are named as Bacmid-V-579, Bacmid-502-510, Bacmid-V-411-417and Bacmid-V-302-309.2. Expression and identification of the chimeric proteinsAll the resulting bacmids were transfected into monolayer Sf9cells with lipofectamine2000as recommended by the manufacturers of the Bac-to-Bac Baculovirus Expression Systems. The cells were harvested at72h post infection (hpi) and washed two times with PBS. The RT-PCR results showed that the mRNA of the four target genes were transcripted in transfected cells. The chimeric proteins, approximately60kDa, were expressed effectively in insect cells and confirmed by SDS-PAGE, Western blot and IFA. The chimeric proteins (vp60-ova/579, vp60-ova/502-510, vp60-ova/411-417and vp60-ova/302-309) could react with both anti-VP60monoclonal antibody (A3C) by Western blot and IFA. In addition, two chimeric proteins (vp60-ova/579and vp60-ova/302-309) still possessed the hemagglutinating activity. However, two other chimeric proteins (vp60-ova/502-510and vp60-ova/411-417) did not have the hemagglutinating activity.3. Electron microscopy and immunogenicity of the chimeric proteinsFour chimeric proteins (vp60-ova/579, Vp60-ova/502-510, vp60-ova/411-417and vp60-ova/302-309) were primary purified and observed by electron microscopy. Then C57BL/6mice were immunized with the purified chimeric proteins, which suspended in PBS without adjuvant, meanwhile setting native RHDV-VLPs, OVA peptides and steriled PBS as control.The titers of VP60-specific antibodies in serum were detected by indirect ELISA at Od,28d and42d. The TNF-α serum concentration were measured by TNF-α instant ELISA kits. The results showed that the chimeric proteins (vp60-ova/579, vp60-ova/502-510, vp60-ova/411-417and vp60-ova/302-309) could correctly assembled into virus-like particles (VLPs) which were morphologically and structurally similar to native RHDV-VLPs. The chemic protein (Vp60-ova/579) could induce more TNF-α secreting and less VP60-specific antibody than other groups. Therefore,579th amino acids of VP60might be a better site to present exogenous T cell epitopes.In conclusion, four chimeric proteins were constructed by integrating the OVA CD8+ T cell epitopes to VP60in this study. And we found that four chimeric RHDV-VLPs can stimulate TNF-a secretion in mice and generate VP60-specific humoral immune response in the absence of adjuvant. All above results shown that the new RHDV-based VLPs might be a suitable system serving as an efficient platform for T cell epitope presentation. The reseach has laid an important theoretical foundation for the possibility of RHDV VLPs as a vector.