VP60 is the main target for new vaccines, as well as for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for controlling RHDV

VP60 is the main target for new vaccines, as well as for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for controlling RHDV. 3F2) that only recognize RHDV1, and four MAbs (1G2, 2C1, 3B7, and 5D6) that only recognize RHDV2 were identified. The epitopes recognized by these MAbs were mapped by Western blotting. Sequence analysis showed that the epitope sequences recognized by 1D6, 1H2, and 3F2 are highly conserved (98%) among RHDV1 strains, whereas the epitope sequences recognized by 1G2, 2C1, 3B7, and 5D6 are 100% conserved among RHDV2 strains. The high conservation of the epitope sequence showed that the screened MAbs were type-specific, and that they could distinguish different RHDV subtypes. Rabbit hemorrhagic disease virus (RHDV) is a member of the family gene, RHDV2 forms a separate branch, which may indicate that RHDV2 is a new number of the genus17. To date, RHDV2 has been detected in Spain, France, Great Britain, and Italy15,20,21,22. At present, RHDV2 has not been reported in Digoxigenin ACAD9 China. Therefore, it is very urgent to study the etiology, epidemiology, diagnosis, and control of RHDV. The hybridoma technique has the potential to provide an inexhaustible supply Digoxigenin of quality monospecific monoclonal antibodies (MAbs)23. MAbs can serve as useful diagnostic tools and act as probes for cellular and macromolecular investigations24. In the present study, the VP60 proteins of RHDV1 and RHDV2 were used as immunogens to prepare RHDV type-specific MAbs by hybridoma fusion. Finally, three MAbs that specifically recognized RHDV1 and four MAbs that specifically recognized RHDV2 were identified. The epitopes recognized by these type-specific MAbs were also precisely mapped. Type-specific MAb preparation provides the foundation for the establishment of RHDV subtype-specific detection methods, and it has important significance for RHDV epidemiological investigations and phylogenetic analyses. Results Expression of recombinant VP60 proteins The entire genes of different RHDV subtypes were expressed as histidine-tagged (His-tagged) fusion proteins in BL21 Star(DE3)pLysS. The VP60 proteins were successfully expressed and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell lysates after induction with isopropyl -D-1-thiogalactopyranoside (IPTG). The VP60 proteins of RHDV1 and RHDV2, pVP60-1 and pVP60-2, respectively, were successfully purified by excising the relevant gel slices and extracting the proteins. Western blotting analysis showed that the recombinant His-tagged VP60 proteins reacted with rabbit hyperimmune serum (for details, see Supplemental Information Figure S1), suggesting that the recombinant VP60 proteins expressed in had good antigenicity and can be used immunogens. The RHDV1 and RHDV2 VP60 proteins were also expressed via a eukaryotic expression system, and they are referred to as sVP60-1 and sVP60-2, respectively. sVP60-1 and sVP60-2 were expressed using the Bac-to-Bac Baculovirus Expression System after recombinant RHDV1 VP60 baculovirus and recombinant RHDV2 VP60 baculovirus were seeded separately onto 9 (Sf9) cells. The genes of RHDV1 and RHDV2 were also cloned into the eukaryotic expression vector pcDNA 3.1(+) to obtain pcDNA-R1-VP60 and pcDNA-R2-VP60, respectively, which were used to separately transfect HeLa cells. The VP60 proteins of RHDV1 and RHDV2 that were expressed Digoxigenin in HeLa cells were named eVP60-1 and eVP60-2, respectively. VP60 expression in Sf9 and HeLa cells was identified by an immunofluorescence assay (IFA). Green fluorescence was detected in Sf9 and HeLa cells after infection or transfection, respectively (for details, see Supplemental Information Figure S2). The results of the IFA suggested that the RHDV1 and RHDV2 VP60 proteins were correctly expressed in Sf9 and HeLa cells. Production of MAbs against recombinant VP60 proteins Female BALB/c mice were separately immunized with purified VP60 proteins from different RHDV subtypes. Hybridomas were obtained by fusing SP2/0 cells with spleen cells from immunized mice. Hybridomas were cultured in 96-well culture plates and subcloned by limiting dilution. Supernatants were assayed 10 days after cell fusion, and wells with.