Development Of Combined Genetic Engineering Vaccine Against Mycoplasma Hyopneumoniae And Porcine Circovirus Type 2

Mycoplasmal pneumoniae of swine(MPS)is a chronic respiratory disease,and also known as swine enzootic pneumonia(EP).The main pathogen of MPS is Mycoplasma hyopneumoniae(Mhp).Porcine circovirus type 2(PCV2)is the main pathogen to induce Post-weaning multisystemic wasting syndrome(PMWS).Clinical co-infections of Mhp with other pathogens,such as PCV2,worsen the severity and potential persistence of related diseases,and eventually leads to an increase in mortality.At present,vaccine immunization is the main means for the prevention and control of Mhp and PCV2 in pigs.However,traditional commercial vaccines are mainly inactivated and attenuated vaccines,which have the deficiency of the poor immunogenicity,unstable immune response duration or easy recovery of virulence.Combined vaccines can prevent multiple diseases in a single dose,especially in the case of long-term co-existence of epidemic diseases.It will become the main vaccine type of future epidemic prevention.Vaccines developed by genetic engineering methods can simultaneously express the antigen proteins of various diseases in order to achieve the effect of combined vaccines.Moreover,the compatibility problems between antigen components of conventional vaccine were also solved.However,at present,there is no report on the combined genetic engineering vaccine which can prevent both Mhp and PCV2.The aim of our study is to simultaneously express chimeric proteins composed of P97R1,P46 and P42 proteins of Mhp and Cap protein of PCV2 on the surface of baculovirus.The recombinant baculovirus rvAc-P97R1P46P42-cap was used as a live virus vaccine,and the immune responses induced in mice and piglets were analyzed.In order to detect the protein expression and the effect of animal immunity,the recombinant proteins of rP97R1,rP46,rP42 and Cap were prepared by prokaryotic expression system,and the antiserum was prepared by immunizing New Zealand rabbits with rP97R1,rP46 and rP42 protein,respectively.The titers of three purified polyclonal antibodies were detected by indirect ELISA,and all of which were more than 1:400000,which could be used in subsequent experiments.Next a recombinant baculovirus with double fluorescent tags named rvAc-P97R1P46P42-mCherry-Cap-EGFP was constructed in this study.After the infection in Sf9 cells,the results from the observations of direct fluorescence,the identifications of indirect immunofluorescence and immunoelectron microscopy show that the P97R1P46P42 chimeric protein fused with the mCherry tag and the Cap protein fused with the EGFP tag can be expressed and displayed along the cell membrane and on the surface of the baculovirus.And then on this basis,the recombinant baculovirus rvAc-P97R1P46P42-cap was obtained by the removal of fluorescence tags.It was used in the immune experiment and its immunogenicity was analyzed.The BALB/c mice were immunized with the recombinant baculovirus rvAc-P97R1P46P42-cap.The detections from indirect ELISA and lymphocyte proliferation assay show that,compared to PBS,rvAc-dual and commercial vaccines,recombinant baculovirus can induce the body of immunized mice to produce specific IgG antibodies(P<0.001),the spleen lymphocyte can produce a significant proliferative effect(P<0.001)after being stimulated by the corresponding stimulus,and the serum can specifically react with the field strains of the two pathogens.These results indicate that this recombinant baculovirus was able to induce specific humoral and cellular immunization in mice.On this basis,similar experiments were carried out on the piglets.ELISA detection of the sera from immunized piglets show that,compared to rvAc-dual,recombinant baculovirus could also induce specific antibodies and cytokines IL-4 and TNF-y(P<0.001)in pigs.It indicates that recombinant baculovirus can also induce specific humoral and cellular immunization in piglets.These results indicate that recombinant baculovirus rvAc-P97R1P46P42-cap have good immunogenicity in mice and piglets.In this study,the chimeric proteins of three Mhp antigens(P97R1,P46 and P42)and the Cap protein of PCV2 were simultaneously expressed through baculovirus surface display system.The immunogenicity of this recombinant baculovirus was confirmed in mice and piglets,which provides a solid basis for the development of combined genetic engineering vaccine to prevent Mhp and PCV2.