| Mycoplasma suis is a pathogen that can cause porcine anemia,It has a high degree of infectivity.The pathogen can cause acute infectious anemia in pigs,creat sow reproductive obstacle,grow slowly.Clinically,the cross infection of the disease and other diseases occurred frequently,which increased the difficulty of disease diagnosis,and brought great difficulties and losed to the development of the pig industry worldwide.The specific primers for this pathogen ENO gene were designed and synthesized according to the open frame of the sequence in GenBank.PCR products were cloned into PMD19T-Simple vector,after double enzyme digestion of Kpn I and Xho I,Gel electrophoresis and gel extraction were carried out and the eukaryotic recombinant expression plasmid PVAX1-ENO was constructed.And then it was transfected into vero cells for expression by lipofectamine 2000.Vero cells were selected for eukaryotic expression of this gene.Microscopic observation of immunofluorescence results showed that cell surface can be observed comparatively complete green fluorescence.Which demonstrated that specific proteins were successfully expressed by the gene.Meanwhile,recombinant adenovirus shuttle plasmid ADV4-ENO,ADV4-ENO and adenovirus backbone plasmids were co-transfected into 293 T cells.Ad5-ENO recombinant adenovirus was successfully constructed.And the Ad5-ENO was amplified and purified.Ad5-ENO recombinant adenovirus vector vaccine was generated.BALB/c mice were immunized with the PVA1-ENO vaccine and the Ad5-ENO adenovirus vaccine by an improved prime-boost strategy.The Ad5-ENO recombinant adenovirus vaccine group,PVAX1-ENO nucleic acid vaccine group,PVAX1 group,and PBS control group were constructed.The mice were sacrificed and serum was collected after vaccine immunization.Ig G,IgG1,Ig G2a antibody levels in serum and IFN-y and IL-4 cytokine levels in serum were measured by ELISA.After the third immunization two weeks later,three mice were randomly selected from each group.The CD4~+and CD8~+in spleen lymphocytes of mice were determined by flow cytometry.The results showed that the PVAX1-ENO nucleic acid vaccine and Ad5-ENO recombinant adenovirus vaccine were successfully constructed,and the recombinant adenovirus titer of Ad5-ENO was 1010PFU/mL.Ig G,IgGl and IgG2a levels of Prime-Boost immunization strategy group,Ad5-EN O vaccine group and PVAX1-ENO nucleic acid vaccine group were significantly higher in serum than PVAX1 empty vector group and PBS control group(P<0.01).IFN-y and IL-4 levels of Prime-Boost immunization strategy group,Ad5-ENO vaccine group and PVAX1-ENO nucleic acid vaccine group were significantly higher than PVAX1 empty vector group and PBS control group(P<0.01).The Lymphocyte subsets CD4~+,CD8~+ analysis showed that the CD4+levels of the Prime-Boost immunization strategy group,Ad5-ENO vaccine group,PVAX1-ENO nucleic acid vaccine group were significantly higher than the PVAX1 empty vector group and PBS control group(P<0.01).There was no significant difference between PVAX1 group and PBS control group(P>0.05).CD8~+levels of the Prime-Boost immunization strategy group were significantly higher than Ad5-ENO vaccine group,PVAX1-ENO nucleic acid vaccine group,PVAX1-ENO nucleic acid vaccine group,PVAX1 empty vector group and PBS control group(P<0.05).There was no significant difference between Ad5-ENO vaccine group,PVAX1-ENO nucleic acid vaccine group,PVAX1 empty vector group and PBS control group(P>0.05).CD4+/CD8+of Prime-Boost immunization strategy group,Ad5-ENO vaccine group and PVAX1-ENO nucleic acid vaccine group was significantly higher than PVAX1 empty vector group and PBS control group(P<0.05).This experiment laid the foundation for the preparation of the new vaccine against Mycoplasma suis and the study of immune strategy. |
