Immune Protective Efficiency Of Recombinant Actinobacillus Pleuropneumonia NLPI Against Chellenge Infection In Mice

Porcine Actinobacillus pleuropneumonia,caused by Actinobacillus pleuropneumoniae(APP),is a very important disease in pig industry.It is one of the major epidemic diseases in the world pig industry.Existing vaccines can not give full protection against APP infection,so it is necessary to screening new vaccines.The pathogenesis of the disease causing the pulmonary lesions,is not clearly understood.We employed a mouse model of intranasal infection by APP,which results in lung inflammation.Results showed that mice showed dyspnea and anorexia after intranasal inoculation with APP at post-inoculation 48 h.In the autopsy,the lungs were found to be severely damaged by acute hemorrhagic pneumonia.The histopathologic changes were characterized by hemorrhage,eosnophils and lymphocyte infiltration.Expression of interleukin-8(IL-8)、IL-10,interferon-alpha(IFN-α)mRNA increased significantly in infected mice lung by APP.and the expression of tumor necrosis factor-alpha(TNF-α),IL-1β,IL-6 mRNA increased very significantly.The Expression of toll-like receptor-2(TLR-2),TLR-4,interferon regulatory factor-1(IRF-1),IRF-5,IRF-7,(NLRP-3)increased significantly compared with control.From these data,we conclude that IL-1β,IL-6,IL-10,INF-α,TNF-α involoved pathogenesis of APP infection.IRF-7 and IRF-5 expression increase driven inflammatory response in mice by self-limiting inflammatory response in mice during APP infection vs TLR-2,4.NLRP-3 inflammasome involved inflammatory response in mice during APP infection.In this study,immune protection of rNLPI vaccination against APP challenged infection was detected.Based on APP nlpi sequences in GenBank,the primers were designed.APP nlpi sequence was obtained by PCR from APP genome.Sequence analysis and function predication was made by bioinformatics software.The results revealed that The length of this gene was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein.Homology analysis showed that it shared high sequence similarity between different serotypes of APP.The APP nlpi was inserted into pET32a(+).Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced byisopropyl β-D-1-thiogalactopyranoside(IPTG)and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and West-blot.The 34 kD-protein was detected and showed good immunogenicity.The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice.The serum immunoglobulin G(IgG)level was assayed by enzyme-linked immune sorbent assay(ELISA).The results showed that 50μg rNLPI + Frund’s incompleted adjuvant,30μg rNLPI+ Frund’s incompleted adjuvant and 10μg rNLPI + Frund’s incompleted adjuvant and 30μg rNLPI respectively induced 41%,22%,10% and 20% survival rate.No mice survived the challenge in adjuvant group and control group.The rest living mice of experiment groups slowly returned to normal health status.From above,rNLPI induced a partial immune protection of mice against APP challenged infection.The study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.