Preparation Of DuCV And DTMUV Virus-like Particles And Evaluation Of Their Immune Effects

Duck circovirus(DuCV)is an infectious disease characterized by immunosuppression.DuCV infection can cause the decrease of immune function and induce the secondary infection of other pathogens.Duck tembusu virus(DTMUV)is an infectious disease caused by Duck tembusu virus(DTMUV).DTMUV can infect many kinds of poultry and birds,especially ducks and geese,the mixed infection of duck circovirus(DuCV)and duck tambucu virus(DTMUV)often occurs in duck industry,which often brings great economic losses to duck industry.There is currently no vaccine to prevent co-infection of the two viruses.In recent years,virus-like particles(VLPs),as one of the most important forms of vaccine development,have attracted much attention.In this study,DTMUV and DuCV virus-like particles were co-expressed by baculovirus system,and their immunogenicity was preliminarily studied,which provided experimental basis for the development of combined vaccine against duck circovirus disease and duck tembusu virus disease.The main contents and findings of the study are as follows:1.Construction of double promoter recombinant plasmidThe Cap gene of DuCV(GH01 strain)and pr ME gene of DTMUV(CQW1 strain)were amplified,and the two target fragments(804 bp and 2037 bp,respectively)were cloned into the downstream p10 and PH promoters of the expression vector p Fast Bac-Dual.The dual promoter expression vector p Fast Bac-Dual-Cap-pr ME was obtained.The recombinant Bacmid-Cap-pr ME was obtained by translocation of the plasmid into DH10Bac receptive cells,and the recombinant Bacmid-cap-pr ME was obtained after blue-white plaque screening.2.Preparation and identification of DuCV and DTMUV VLPsThe constructed Bacmid-Cap-pr ME recombinant plasmid was transfected into Sf9cells,and cultured at 28℃for 72 h,supernatant was collected to obtain P1 generation recombinant baculovirus(named r Bac-Cap-pr ME).The recombinant baculovirus was transmitted to P4 generation,and cells were collected for protein Western blot.WB),the results showed specific bands between 25-35 k Da and 55-70 k Da,indicating that Cap and pr ME proteins were successfully expressed in Sf9 cells.Indirect immunofluorescence assay of Sf9 cells infected with recombinant baculovirus virus showed that red fluorescence could be detected in Sf9 cells incubated with either DuCV Cap or DTMUV E polyclonal antibodies.These results indicated that the target protein could be expressed in Sf9 cells.A large number of particles with diameters of 15 nm and 50 nm were found in Sf9 cells infected with recombinant baculovirus(r Bac-Cap-pr ME)by electron microscopy,indicating that both Cap and pr ME proteins could self-assemble to form VLPs in Sf9 cells.3.Preliminary study on the immunogenicity of DuCV and DTMUV VLPsVLPs vaccine was prepared by mixing DuCV and DTMUV VLPs with Freund adjuvant and emulsifying.The ducks were injected with the vaccine and divided into the following groups:VLPs group;Inactivated vaccine group;PBS group.The first exemption was given at 7 d of age,2 at 21 d and 3 at 35 d.Serum and peripheral blood were collected at 7d,14 d,21 d,28 d,35 d,42 d,49 d and 56 d after first immunization.The serum Ig G antibody levels collected at the above time points were detected.The results showed that specific Ig G antibody could be produced in the VLPs vaccine group after the first immunization,and the Ig G antibody level in the VLPs vaccine group reached the peak(OD450nm=0.85)at 42 days,and was significantly different from the inactivated vaccine group(P<0.05).Neutralizing antibody was detected in serum of ducks at 4 and 6 weeks after first immunization.The results showed that specific neutralizing antibody could be produced in both VLPs vaccine group and inactivated vaccine group,and the difference was statistically significant compared with PBS group(P<0.05).The expression levels of CD4~+,CD8~+IL-4 and IFN-γin peripheral blood at 1 w,3 w,5 w and 7 w were detected by fluorescence quantification.The results showed that the contents of CD4~+,CD8~+,IL-4 and IFN-γin peripheral blood of VLPs vaccine group and inactivated vaccine group were higher than those of PBS group(P<0.05).These results indicate that VLPs vaccine can stimulate humoral immunity and cellular immunity.4.DuCV and DTMUV VLPs protection testsThe animal challenge protection test was conducted at 3 w after the third immunization,and the results showed that the liver,spleen and brain tissue of ducks in the VLPs vaccine group and inactivated vaccine group did not show obvious lesions,while the splenic stasis of ducks in the PBS group was enlarged,the edges were blunt,and the brain tissue bleeding was obvious.The virus copy number in liver,spleen and brain tissues in PBS group was significantly lower than that in VLPs vaccine group(P<0.05).Histopathologic observation showed that there were no obvious lesions in liver,spleen and brain tissue of VLPs vaccine group and inactivated vaccine group.However,in PBS group,liver bleeding,hepatocyte steatosis and hepatic cord arrangement disorder occurred after the challenge.In spleen,the boundary between white pulp and red pulp was not clear and lymphocyte infiltration was observed.Brain tissue vascular"cuff"lesions.In conclusion,the double promoter expression vector p Fast Bac-Dual-Cap-pr ME can co-express DuCV Cap protein and DTMUV pr ME protein in baculovirus expression system,the two proteins could self-assemble into virus-like particles in Sf9 cells.The VLPs vaccine can induce humoral and cellular immunity,effectively resist virus infection,reduce the damage of virus to tissues and organs,and provide a strong protective effect for the body.