| Rabbit hemorrhagic disease virus(RHDV) causes rabbit hemorrhagic disease(RHD), which is characterized by70-100% mortality rates in adult rabbits. RHDV infection distributed worldwide and caused a significant economic loss to rabbit production. Vaccination remains the most effective method for the prevention of RHD. Though some subunit vaccines against RHDV have been made great progress recent years, the commercially available vaccines are still traditional inactivated vaccines due to the high costs and low expression productions. The VP60 protein which is the major structural protein has the ability to be self-assembled into virus-like particles(VLPs). Here, we successfully produced RHDV VLPs derived from recombinant VP60 protein expressed in E.coli using a SUMO tag to promoting soluble expression. Subsequently, we proved that the VLPs might be a potential desire for developing the submit vaccine against RHDV. And then, BALB/c mice were inoculated with VLPs to obtain monoclonal antibody against VP60. At last, a rapid、simple and high specificity double antibody-sandwich enzyme-linked immunosorbent assay(DAS-ELISA) method was established using monoclonal antibody against VP60.In this study, VP60 gene was inserted into a pSMK expression vector containing a small ubiquitin-like modifier(SUMO) tag which can promote the soluble expression of heterologous proteins in E. coli cells and transformed into BL21-condon Plus(DE3)-RIL cells. The recombinant VP60 protein was soluble expression in E.coli and was further confirmed with SDS-PAGE and western blot assay.After digested using SUMO protease, the purified recombinant VP60 protein was purified again. The observing results of electron microscope showed that the VP60 protein without the SUMO tag could self-assemble into VLPs with a diameter of 25-30 nm, and its shape was similar to the authentic RHDV. In addition, the results of western blot indicated that the VLPs could be recognized by rabbit anti-VP60 positive polyclone serum.To evaluate the immunity of RHDV VLPs, rabbits were inoculated with inactivated vaccine, recombinant VP60, VLPs and PBS, respectively. The serum samples were collected at a 7-day interval. The specific anti-VP60 antibodies were detected using an indirect ELISA method and the results showed that the level of serum antibodies increased significantly in both VLPs group and inactivated vaccine groups. Besides, RHDV VLPs also induced significant lymphocyte proliferative responses. The mean level of IFN-γ and IL-4 in VLPs group was higher than PBS group. All of the results suggested that RHDV VLPs could be a promising RHDV vaccine candidate.Because RHDV VLPs showed good immunogenicity, we hope to prepare some specific monoclonal antibodies against RHDV with the VLPs. Firstly, BALB/c mice were inoculated with the purified VLPs, and then mouse spleen cells were fused with SP2/0 cells. At last, seven positive hybridomas were obtained and named as 4A1, 5B2, 6F6, 9H9, 11D4, 15C3, 16F2. Western blot results showed that 7 Mabs against VP60 could reacted to recombinant VP60 protein above. IFA results showed that all the 7 strains Mabs against VP60 could reacted VP60 protein expressed in BHK-21 cell. The Pairing of monoclonal antibodies assay confirmed 9H9 as the optimal capturing antibody and HRP-9H9 as detecting antibodies in a double antibody-sandwich enzyme-linked immunosorbent assay for detection of RHDV antigens. The monoclonal antibody could improve the specificity of the test results. The positive-detection rate of 12 RHDV samples was 100%. In clinical test, 85 samples isolated from rabbits were subjected to DAS-ELISA and RTPCR. The overall agreement between the results obtained by DAS-ELISA and PCR was 98.8%. These data demonstrated that the established DAS-ELISA showed highly specific and sufficiently sensitive for the detection of RHDV. |
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