| The rabbit Bordetella bronchiseptica disease is a chronic respiratory disease in rabbits caused by the Bordetella Bronchiseptica (Bb), which is also described as long-lasts, spread widely, and difficult to cure, resulting in great economic losses to the rabbit industry. Currently protective prevention of the disease is whole-cell inactivated vaccine, which couldn’t offer efficient protection against Bb infections in rabbits. Developing new vaccine candidate components for diagnosis and prevention of the disease is necessary. Outer membrane proteins (OMPs) played an important role in drug resistance, pathogenicity and immunogenicity.This study is based on immunoproteomic analysis of B.bronchiseptica OMPs. Five newly discovered immunogenic proteins were selected as target proteins by bioinformatics analysis, cloned and recombinantly expressed.,and then immune responses of mice to recombinant proteins vaccination were detected, recombinant outer membrane porin protein precursor and putative lipoprotein showed protective indices against challenges with Bb. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for Bordetella bronchiseptica.1 Cloning and sequence analysis of the target geneFive immunogenic proteins (putative amino acid ABC transporter solute-binding protein, ABC; leu/ile/val-binding protein precursor, BPP; conserved hypothetical protein, CHP; putative lipoprotein, PL; outer membrane porin protein precursor, PPP) were selected as the target protein through bioinformatic analysis according to preliminary immunoproteomics assay results of outer membrane proteins of Bordetella bronchiseptica. Five pairs of specific primers were designed by Oligo6.24 software refered to the published GeneBank gene sequence of the target protein, full-length sequence of the target gene was amplified and connected to pMD-18 T vector, Positive plasmid DNA was extracted by alkaline lysis method, and identifid by double digestion and sequencing. Sequence analysis showed that homology of the amplified genes and the reference sequences reached more than 97%, which were the target nucleic acid fragments encoding open reading frames.2 Prokaryotic expression of the target proteins and immunogenicity analysisThe purified PCR products were cloned into pET-28a (+), the plasmids were transformed into E. coli Rosetta (DE3). The plasmids were verified by double enzyme digestion and DNA sequencing. After induced by IPTG, five target proteins were successfully expressed, stripe size:ABC protein is 56.6KDa, BPP protein is 39.7 KDa, CHP protein is 21.6 KDa, PL protein is 41.5 KDa, PPP protein is 41.3 KDa, which were matched with the expected size Western-blot analysis showed that all of the obtained five recombinant proteins could be recognized by the positive serum of rabbit B.bronchiseptica.3 Evaluation of immune efficacy of recombinant proteinsThe ICR mice were inoculated with the recombinant proteins in Freund’s complete adjuvant for evaluation of immune effect of recombinant proteins, nine mice each group, immunized twice,2 weeks interval, piror immunization and challenge, Sera of five randomly selected mice were taken, antibody titer were detected by indirect ELISA method. 2 weeks after the second immunization, eight mice taken from each group were challenged with a concentration of 1.74*107cfu/ml of B.bronchiseptica intraperitoneally. The results showed that the recombinant proteins induced high titer IgG antibody, PPP protein and PL protein provided better protection, protection ratio of mice was 62.5% and 50% respectively compared to 12.5% of blank controls. Protection Ratio of ABC protein, BPP protein and CHP protein had little difference with the control group.For the acquired immune protective proteins PPP protein and PL protein, further test was conducted to determine antibody subtype. The results showed the mice immunized recombined PPP protein produced high titre of specific IgGl and IgG2a antibodies, indicating that the PPP protein can stimulate the body to produce humoral immune response and cellular immune response, but the specific IgG1 antibody titre was higher than specific IgG2a antibody, indicating that PPP protein induced Th2-type dominated immune response (IgG1:IgG2a>1). Mice immunized with PL protein and whole-cell protein mainly produced high titre IgG1 antibodies, but low titre IgG2a antibodies, indicating that PL proteins and whole-cell protein mainly stimulated humoral immune response, which were also Th2-type dominated (IgG1:IgG2a> 1). Spleens were taken respectively from mice immunized PPP protein, PL protein and whole cell protein, spleen lymphocytes were isolated to be used in lymphocyte proliferation assay, IFN-γ, IL-2, IL-4, IL-10 levels in lymphocyte supernatant was detected by commercial detection kit. Levels of IFN-γ, IL-2, IL-4 were significantly increased after stimulated by PP protein and PL protein, while increase in whole-cell protein group was not obvious; PL protein enhanced IL-10 production, while PPP protein and whole-cell protein group had no significant increase. Test of antibody subtype classification proved that PPP protein and PL protein induced a higher humoral immune response than cell-mediated immune response in mice, while PPP protein can also induce certain cell-mediated immune response in mice. Lymphocyte proliferation assays showed that PPP protein and PL protein can effectively stimulate lymphocyte proliferation. Detection of cytokines showed that PPP protein and PL protein can induce both humoral immune response and cell-mediated immune response in mice. |
PDF Full Text Request