The Study On The Component And Immunogenicity Of Haemophilus Parasuis Outer Membrane Vesicles

Haemophilus parasuis(HPS),which belongs to the Pasteur Enterobacteriaceae Haemophilus, is a kind of pleomorphic Gram-negative bacteria. This symbiotic bacteria exists in the upper respiratory tract of pigs. HPS causes a high mortality of porcine Buerger’s disease, of which the main feature is polyserositis, arthritis and meningitis. HPS was identified with 15 serum type, while some can’t be stereotyped. The strains are lack of cross protective immunity, through the virulence factors and pathogenic mechanism have been done a lot of research, developing new effective HPS vaccine is still a urgent problem of our swine disease prevention.In the stable stage, the gram negative bacteria will release the outer membrane vesicles(OMV) to the peripheral environment, which includes outer membrane protein and lipopolysaccharide(LPS) with good immunogenicity. Therefore, OMV is one possible virulence factors of HPS, science OMV contains major attachment proteins--Omp P5, so OMVs are relate to bacterial colonization in the body. The antiserum of Omp and the antigen of serotype HPS are cross reactive, so OMV may be a kind of protective antigen.To understand the composition and immunogenicity of HPS serotype 5 strain OMV, we use the methods of density gradient centrifugation and centrifugation to extract and purify HPS OMV.The major protein components of purified OMV were analysed by SDS-PAGE, and compared with the cytoplasmic protein(CP), periplasmic protein(PP) and outer membrane protein OMP contained by the same strain at the same time; the protein contents of OMV were tested by BCA method, which calculated the extraction efficiency of OMV; we use Pico Green method to determine DNA concentration of OMV. In the animal experiment, we use the different dosage of purified OMV immunize female CD1 mice, the levels of antibody in serum checked by indirect ELISA test, then we analyse the protection rate of statistical at the chickens of virulent strains, which confirmed the immunogenicity and protective effect; the purified OMV was digested by proteinase K, then inoculated in HEP2 cells. The determination of IL-8 content in supernatant, confirmed the immune regulation effect of cytokines.SDS-PAGE results showed that the major protein component of OMP and OMV are similar, and the content of protein was mainly distributed in 32~57 k D; DNA concentration determination showed that OMV DNA was mainly located in the cavity, and the content is much more than the vesicle surface;The challenge test and ELISA confirm that OMV have immune protective effect after the mice immunization, and the valence is stability; IL-8 test results demonstrated that OMV regulates cellular factor. The biological characteristics research shows that, OMV has a dynamic role in the pathogenesis of HPS, which provides a basis for the research of HPS OMV vaccine.