Preparation Of FRBD-Ferritin Nanoparticle Vaccine Based On FIPV Receptor-Binding Domain

Feline infectious peritonitis virus(FIPV)belongs to the genus of the Coronavirus family alphacoronavirus,a type of Feline Coronavirus(FCo V),which can cause Feline Infectious Peritonitis(FIP),a disease with a high fatality rate caused by an abnormal immune response in cats.FIP is one of the most important fatal infectious diseases in cats,and its pathogenesis has not yet been fully revealed.Although there is no direct evidence that FIPV is highly contagious,the common FCo V may mutate to FIPV,and FCo V is the virus with the highest infection rate in cats and has high contagion.Although a variety of drugs have been developed clinically for the treatment of FIP,the effect is not obvious.At the same time,none of the FIPV vaccines developed by conventional technologies can effectively protect the host,and even trigger the antibody-dependent enhancement(ADE)of FIPV infection.Therefore,the design and production of a safe and efficient FIPV vaccine is very important for the prevention and treatment of the disease.FIPV relies on the spike(S)protein that forms the viral envelope for receptor recognition and completes the adsorption and invasion of host cells.Therefore,this study is based on the FIPV S protein and cell receptor binding domain(Receptor binding domain,RBD)to design a vaccine.In order to improve the stability and immunogenicity of the vaccine,ferritin nanoparticles were selected as the antigen presentation system.After ferritin is expressed,it can self-assemble into a nanocage structure,which provides a good platform for RBD display,activate the host to produce neutralizing antibodies that can block the binding of S protein to host receptors,and resist FIPV infection.First,we constructed a recombinant plasmid containing both the FIPV RBD domain and Ferritin gene,transformed it into a shuttle plasmid and transfected SF9 insect cells,and successfully rescued a recombinant baculovirus that can express f RBD-Ferritin self-assembled protein nanoparticles.Subsequently,through PCR,WB and IFA identification,it was determined that the recombinant baculovirus can express the f RBD-Ferritin protein in a secreted form after infecting SF9 insect cells.The protein expression level is the highest at 84 h after infection,and the virus can be passaged stably.At the same time,the results of electron microscopy showed that after the fusion expression of f RBD and Ferritin protein,the nanocage structure formed by Ferritin formed a good display effect on f RBD protein.In order to evaluate whether the f RBD-Ferritin self-assembled protein nanoparticle vaccine can induce the body to produce specific antibodies against FIPV,a vaccine immunization experiment was carried out on mice.The results show that after vaccine immunization,mice can produce specific antibodies against FIPV RBD,and the antibody level lasts for a long time.Finally,we prepared a pseudovirus based on the FIPV S gene,and verified that the specific antibodies produced after vaccine immunization have the ability to neutralize the FIPV pseudovirus.In summary,the recombinant baculovirus rescued in this study can efficiently express f RBD-Ferritin self-assembled protein nanoparticles,and can produce specific antibodies with neutralizing ability to the virus after immunization.It provides a new candidate vaccine for the prevention and control of FIPV.