Study On Multicomponent Recombinant Subunit Vaccines Against Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia(PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae(APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia in pigs. Since this disease was discovered in 1957, it spread in the worldwide and caused important economic losses in industrialized pig production. Because APP has resistance, vaccination has been a main measure to prevent PCP. At present, an inactivated whole cell vaccine is mostly applied for PCP prevention and treatment in China. But APP has 15 serotypes, so the protection efficacy of inactivated vaccines is unfavorable and developing novel vaccine has become a research hotspot. It had been proved that many attenuated vaccines constructed by molecular biology methods and new ghosts vaccine could provide effective protection . But along with the declining virulence of attenuated vaccines, the immunogenicity was also decreased, and moreover, the reversion to virulence might be presented, and the ghost vaccine might be inactivated incompletely.Therefore, subunit vaccines containing extracting natural organism or its secretion had no those disadvantage and could provide effective protection. So, subunit vaccines become an ideal research direction. But if using the component of natural organism or secretion as the component of subunit vaccine, the cost of extraction is high and the production is low. So, applying recombinant technique, six toxic factors of APP were expressed, then constructed two multi-components recombinant subunit vaccines and assessed their protective efficacy in this study.It had been proved that ApxI, ApxII , ApxIII and OMP was more important toxic factors and immunogen, which had been studied as vaccine component. But ApfA and ApxIV that were more toxic facors had been not studied as vaccine component.However, ApfA was proved to play an important role in the course of its residing in host cells and ApxIV could stimulate body to produce higher level of antibodies. Therefore, it was presumed that ApfA and ApxIV might facilitate cross-protection. So, in this study, ApfA and ApxIV was studied as vaccine component firstly. And two multi-components recombinant subunit vaccines were constructed firstly, with one containing 4 recombinant proteins, the other containing 6 recombinant ptoteins. Through the test of immuno-protection against challenge in BALB/c mice, the protection efficacy of multi-component recombinant subunit vaccine was compared with inactive vaccine.1 The apxⅢA, apxIVA and omp gene were amplified from APP serotype 2 strain S1536, APP serotype 1 strain 72-1 and APP serotype 7 strain 25-4 respectively by PCR. The apxⅢA, apxIVA and omp gene sequence was analyzed by homology comparing with others published in GenBank. The results showed that the apxⅢA gene shared 98.4% and 97.8% in nucleotide homology with APP serotype 2 and serotype 8 strain 405 respectively, and the apxIVA gene shared 99.9%和99.2% in nucleotide homology with APP serotype 1 strain 4074 and serotype 3 strain HV114 respectively,...