Construction And Immunogenicity Analysis Of American Type PRRSV Candidate Vaccine Based On Hepatitis B Virus Core Protein

At present, PRRS is prevailing all over the world, and the virus shows high heterogeneity, imposing a great barrier on preventing and controlling disease. At this stage, vaccination is the most important measure for preventing PRRS, but it has its limitations and existing vaccines cannot provide swine with overall. Therefore, it is meaningful to develop new vaccine to prevent and control PRRSV.Since the first successful effort to express the epitope of hand-foot-and-mouth disease which was fused to hepatitis B virus core(HBc), HBc had been increasingly applied to the research on the vaccine of virus-like particles(VLPs). As a carrier protein of VLPs, HBc has a number of characteristic, such as efficient self-assembly into particles under natural conditions, flexible insertional locus, high immunogenicity(over 95%) and no cytotoxicity, presenting antigen, stable expression in variety of systems and etc. In this study, HBc was used as a carrier protein to express GP5 of PRRSV, and to construct a new VLPs vaccine. Then BALB/c mice were immune to evaluate the immunogenicity of recombinant HBc particles(p EO-LEORF).At first, the HBc sequence has been codon optimized, which was based on the codon bias of Escherichia coli(strain K12).The HBc gene was successfully cloned into the genetically modified p ET-28 a and to gain recombinant prokaryotic expression plasmid p EO-NEW. SDS-PAGE, Western blot and the transmission electron microscope were utilized to analyze the character and structure of the recombinant plasmid. Results showed that the HBc was successfully induced to express, and the objective protein can self-assemble into VLPs. It provided a foundation for the further research of recombinant vaccine.The GP5 gene was inserted into the MIR of p EO-NEW, and to gain recombinant plasmid p EO-LEORF(GP5-HBc-based VLPs). The expressed protein was detected by SDS-PAGE and Western blot. The results demonstrated that GP5 protein was successfully expressed and has immunogenicity. The transmission electron microscope was used to observe the morphology of p EO-LEORF and the result showed that recombinant protein can self-assemble into VLPs.The BALB/c mice were intramuscular injection 50 μg p EO-NEW and p EO-LEORF with adjuvant to analyze immunization of the GP5-HBc-based VLPs candidate vaccine. The result of ELISA showed that this vaccine could induce the body to produce specific antibody; by assaying the number of T cell subsets and the level of cytokines, we found that the vaccine groups were significantly higher than control group on CD3+CD4+T cell subset of splenic lymphocytes and IL-4 level(P<0.05). Compared with control group, vaccine groups improved significantly on lymphocyte stimulation index(SI) of splenic lymphocytes and IFN-γ level after the third immunization(P<0.05). All results showed that GP5-HBc-based VLPs candidate vaccine could simulate VLPs, and present GP5 to induce the body to produce humoral and cellular immunity.This study provided data basis for the next step research.