Effect Of Haemophilus Parasuis Outer Membrane Proteins P5 On The Maturation Of Dendritic Cell In Guinea Pigs And Property Research

Haemophilus parasuis, a small pleomorphic gram-negative rod of the family Pasteurellaceae, normally colonize the upper respiratory tract of pigs which constitute their natural reservoir. The bacteria is the etiological agent of porcine Glasser's disease in pigs which is characterized by fibrinous polyserositis, arthritis and meningitis producig significant morbidity and mortality in piglets in pig farms. There are 15 characterized Haemophilus parasuis serotypes, and minimal cross-protection is observed between strains. Virulence factors involved in Haemophilus parasuis infection and pathogenesis are still unknown.The outer membrane proteins (OMPs)of Haemophilus parasuis may contribute to immunity and virulence. The P5 protein is one of the major outer membrane proteins of Haemophilus parasuis, is a major pathogen adhesion protein concerned with the colonization of bacteria. From these content inference the P5 maybe as a novel candidate antigen against Haemophilus parasuis challenged. Therefore expression of Hps OMP P5 gene, purify the P5 and effect of P5 on the dendritic cell in Guinea pigs; Guinea pigs were immunized with P5 and were challenged with Haemophilus parasuis to investigate the ability as a vaccines candidate. The main experiments and results are as follows:1. Naive DC were cultured with GM-CSF and IL-4, and then trigged DC maturation with different concentration protein P5, the related cytokine during P5 stimulation like IL-6, TNF-α, IL-12p70 were detected. Protein P5 can stimulate DC maturation. The concentration of IL-6 and TNF-a apparent higher compared with blank control group when DC stimulated by 5μg/ml P5(P<0.01), IL-12 sight increasing conpared with blank control group(P>0.05). The concentration of IL-6 is higher than control group when DC were stimulated by 50μg/ml P5(P<0.01), but IL-12p70 and TNF-αwere lower compare with blank control group(P<0.01). The Triggered DC with protein P5 has a improved Promotive effect to T lymphocyte proliferation(P<0.01). (Conclusion)Protein P5 can promote maturation and proliferation of bone marrow-derived DC and improve the Promotive effect to T lymphocyte proliferation and function.2. The recombinant outer membrane proteins P5 of Haemophilus parasuis, mixed and emulsified 1:1 with Freund's adjuvant, immune guinea pigs, meanwhile set Hps whole-cell inactivated vaccine as positive control, and sterile PBS as negative control. The antibodies against P5 were detected by indirect ELISA, the proliferative response of spleen lymphocytes of the guinea pigs to P5 was observed by MTT assay, and injecting to the abdominal cavity of the immunned guinea pigs with 2×LD50 Hps. The results indicated that the vaccine carrying the outer membrane protein P5 of haemophilus parasuis was able to elicit strong specific humoral immuned responses and cellular immuned responses in guinea pigs. Protective efficacy results demonstrated that survival rate of protein immune group against serotype 4 and serotype 12 are 17%(1/6), against serotype 5 is 50%(3/6), all better than the control(0/6), the survival rate of positive control group is 50%.This research sufficient DC generated from bone marrow cells are induced in vitro and strongly stimulate proliferation of T cells after Protein P5 are loaded. P5 seems to have a potent immunogenicity and induces protection against haemophilus parasuis. It can provide important theoretical bases for the Hps OMP P5 used as a novel vaccine candidate protein.