Immune Efficacy Of The Major Protective Antigens Of Haemophilus Parasuis

Haemophilus parasuis (HPS) is one of the colonizers as the normal microflora of porcineupper respiratory tract, but it is also the etiological agent of Glasser’s disease,which ischaracterized as fibrinous polyserositis, arthritis and meningitis in veterinary clinics.HPS hasdiversities of serotypes and large proportion of non-typeable strain. However, the currentcommercial vaccines can notafford effective protections because of low cross protection amongdifferent serotypes, as well as a lack of understanding of the virulence factors and protectiveantigens of this bacterium. Monovalent and multivalent inactivated vaccine is mainly used forthe prevention and treatment of HPS, but the effect is not ideal.It has been reported, subunitvaccines can produce a certain degree of immune protection effect. Therefore, the purpose of thisstudy was to assess Immune Efficacy of the Major Protective Antigens of HPS and lay afoundation for research and development ofsubunitvaccine.Trimeric autotransporters (VtaA) are virulence associated proteins of HPS. There are13copies of the vtaA gene in HPS serotype5Nagasaki strain genome which vtaA1genes and othercopies have high homology.The sequences encoding N-terminal (VtaA1N), C-terminal (VtaA1C)and the middle section (VtaA1M) of the VtaA1was expressed. The immunoproteomics methodswere used to identify two antigen protein-HktE(catalase) and AfuA (Fe3+transporter). Thesequences were amplified by PCR from genomic DNA of H.parasuis serotype5Nagasakistrainand cloned into pET-28a(+) to construct the recombinant plasmids of pET-afuA, pET-hktE,pET-vtaA1C,pET-vtaA1M and pET-vtaA1N for expressions in E.coli BL21(DE3). Afterinduction by IPTG,optimization of reaction temperature and reaction conditions, express thetarget protein, which the results consistent with the expected results in which HktE and AfuAexpressed in soluble form, VtaA1C, VtaA1M, VtaA1N expressed in the form of inclusion bodies.Ni-MCAC was used to purify recombinant proteins. Due to the low expression level of VtaA1Nit could not be purified.The remaining four proteins were obtained by this method with highpurity protein. Western blot analysis showed that the recombinant protein has a goodreactogenicity. The recombinant protein AfuA, HktE,VtaA1C,VtaA1M which has been purified and theremaining six kinds of protein OppA, HbpA, HPS-04307, CdtA, CdtB, CdtC which haspreviously been constructed and purified in the laboratory were use to immunized4-6weeks KMmale mice with100μg.15d after the second immunization Antibody titers were determined byindirect ELISA and detection of cytokines. MTT assay was applyed in mice spleen lymphocyteproliferation.KM mice were intraperitoneal challenged with a lethal dose of5LD50of HPSserotype5and13Nagasaki strain. Immunization and challenge assay demonstrated all the10recombinant proteins could induced high levels of antibodies. Lymphocyte proliferanon assayresults that the group can have a significant cell-mediated immunity, IL-2, IL-4, IFN-γweresignificantly increased after the immunization. Which OppA, CdtC, CdtA, CdtB, AfuA, HktE,VtaA1C, VtaA1M protein can provide higher protection and has the potential as candidates forantigen protein.This study hopes to lay the foundation for the development of subunit vaccines.