Preparation And Immunogenicity Of PRRSV Virus-like Particles

Porcine reproductive and respiratory syndrome virus(PRRSV)can cause reproductive failure in pregnant sows and respiratory problems in piglets.In recent years,a strain with high gene homology with the NADC30 strain has been discovered in my country,named NADC30-like PRRSV.There is currently no commercial vaccine for the prevention of NADC30-like PRRSV.Virus-like particles have inherent immunogenicity,can induce cellular and humoral immunity,and because they do not contain viral genetic material,they have high safety characteristics,so they play an important role in vaccine development.Therefore,this study will develop a virus-like particle(VLPs)vaccine against NADC30-like PRRSV.Construction and identification of virus-like particles(VLPs)of NADC30-like PRRSV.In this experiment,the shuttle plasmids p FB-30-ORF5,p FB-30-ORF6 and p FB-30-ORF5-ORF6 were first constructed for the GP5 and M proteins of the PRRSV SD-A19 strain.They were respectively transformed into Escherichia coli DH10 Bac competent cells to obtain three kinds of recombinant bacmids,which were transfected into SF9 insect cells respectively.When the cells were obviously diseased,the recombinant baculovirus liquid was harvested and blindly passed to the third generation.After infecting the adherent SF9 cells with the third-generation virus liquid,the recombinant proteins were identified by Western blot,immunoelectron microscopy,and indirect immunofluorescence.The results showed that three kinds of recombinant baculoviruses were successfully packaged,and three kinds of VLPs of GP5,M and GP5-M were prepared by packaging.After the successful preparation of VLPs,optimize the optimal expression conditions of VLPs,and purify by sucrose gradient centrifugation after massive expression of VLPs.Prokaryotic expression of NADC30-like PRRSV GP5 protein.In order to express the GP5 protein of SD-A19 strain,the prokaryotic vectors p ET-30a(+)-30-GP5 and p ColdⅡ-30-GP5 were constructed and transformed into E.coli BL21 competent cells.The induction conditions were optimized and analyzed by SDS-PAGE and Western blot.The strains verified to be correct were cultured in large quantities,protein samples were prepared,purified by a nickel column,the urea concentration in the eluate was optimized,analyzed by SDS-PAGE,and the gel was scanned by thin-layer chromatography.The results showed that the GP5 protein was successfully expressed by two prokaryotic expression plasmids and purified by nickel column.The purity of GP5 protein was 63.3%.Animal immune evaluation of NADC30-like PRRSV VLPs.Mice and piglets were immunized with the three VLPs at different doses and with commercial inactivated vaccine and PBS as controls.Peripheral blood was collected weekly and serum was separated.The immunogenicity of VLPs was judged by various immune index tests and challenge protection experiments.The results showed that:The immunization process did not cause animal death;All the three VLPs could stimulate the cellular and humoral immunity of mice and piglets,and increase the level of GP5 specific antibody and neutralizing antibody.All of the three VLPs can effectively reduce the viral load in the lung,so the three VLPs constructed in this experiment can resist the influence of PRRSV on pigs to a certain extent,which provides the possibility for the development of commercial PRRSV VLP vaccine.