Genome-wide Identification Of Immunoprotective Lipoproteins In Actinobacillus Pleuropneumoniae

Porcine pleuropneumonia is a highly contagious respiratory disease caused by Actinobacillus pleuropneumoniae?A.pleuropneumoniae?.A.pleuropneumoniae has 18 serotypes and many virulence factors.The Currently vaccines against porcine pleuropneumonia on the market are inactivated vaccines and subunit vaccines.Though they have positive efforts on the control of this disease,they have some defects:the insufficiency of inactivated vaccines,limited immunoprotective factors of subunit vaccines.They cannot completely exclude bacterial colonization and persistent infection.Therefore,it is necessary to further understand the pathogenesis of A.pleuropneumoniae and explore antigens with potential for immunoprotection,in order to develop more effective vaccines.Many immunogenic lipoprotein in the outer membrane of A.pleuropneumoniaeis conserved among various serotypes,and are considered as potential vaccine candidates.This study aims to find immunoprotective lipoproteins of A.pleuropneumoniae,so as develop effective subunit vaccines.The research process and main results are described briefly as follows:1.Primer design for lipoproteins cloning:In the previous study,60 possible lipoproteins were screened from A.pleuropneumoniae strain JL03?serotype 3?by bioinformatics methods,The biological properties of lipoprotein Lip40 and PalA were analyzed before.Therefore,this study will analyze the immunological activity and immunoprotection of the remaining 58 lipoproteins in detail.After sequence analysis,primers for lipoprotein cloning expression were designed by rationally removing the lipoprotein signal peptide sequence and introducing appropriate restriction sites.2.Construction of prokaryotic expression vector of lipoprotein:58 lipoprotein genes were amplified by PCR from A.pleuropneumoniae JL03 genome.PCR products were ligated into pMD18-T vector and transformed into E.coli DH5a.The target fragments were then digested using proper restriction enzymes and inserted into pGEX-KG to construct expression plasmids.A total of 55 recombinant plasmids were successfully constructed.3.Expression,purification of recombinant lipoprotein and western blot analysis:Expression plasmids were transformed into E.coli BL21.After IPTG induction,the overexpression of recombinant lipoproteins were analyzed by SDS-PAGE.A total of 47 lipoproteins were successfully expressed,thirty-seven of which were expressed in the supernatant of E.Coli BL21 lysate and 10 were insoluble.Soluble proteins were purified by affinity chromatography and subjected to immunoblot analysis.Five representative lipoproteins with relatively strong signals in western blot were used in the animal experiments,including APJL₀386,APJL₀922,APJL₁380,APJL₁740 and APJL₁976.4.Immunoprotective identification of representative lipoproteins in mice:The 5 selected lipoproteins were used as immunogens.BALB/c mice were immunized twice.Mice were challenged with A.pleuropneumoniae serotype 1 strain 4074?serovar 1?two weeks after boost immunization.The humoral immune responses of mice were detected by ELISA and Western blot assays.It was found that each group of immunogens could elicit a specific immune response.The survival rates of APJL₁380,APJL₁976,APJL₀922,APJL₀386,and APJL₁740 were 92%,75%,75%,25%,and 42%,respectively.Lung sections showed acute hemorrhagic pneumonia,while mice survived from the infection in the immunized groups showed healthy lung sections.Groups of BALB/c mice were passively immunized with the serum obtained from the active immunization,and then challenged with letal A.pleuropneumoniae strain 4074,The survival rates in the groups immunized with antiserum against APJL₁380,APJL₁976,APJL₀922,APJL₀386 and APJL₁740 were 83%,92%,67%,33%and 25%,respectively.No mice immunized with antisera against GST and PBS was survived after challenge.Survived mice immunized with antiserum against lipoproteins showed healthy lung sections,while mice immunized with anti-GST and PBS sera showed severe lung lesions under microscopic analysis.Three lipoproteins,APJL₀922,APJL₁380 and APJL₁976 were found from A.pleuropneumoniae in this study.They showed good immunogenicity,can induce high-level humoral immune responses,and protect animals from lethal bacterial challenge.These proteins are expected to be used to develop high-efficiency subunit vaccines against A.pleuropneumoniae infection.