| Objectives: Leptospirosis, caused by the pathogenic leptospires, is one of the most widespread zoonotic diseases as known. Currently, the commercially available vaccines against leptospirosis only produce short-term immunity and provide no cross-protection against the different serovars of pathogenic leptospira. Outer membrane proteins of leptospira play an important role on the infection and cross-protection, so OMPs have become a major focus of leptospirosis research. To find the new potential efficacious leptospiral vaccine genes, we analyzed the whole sequences of L. interrogans serogroup icterohaemorrhagiae serovar Lai strain #56601 and identified the immunity of them by reverse vaccinology technology.Methods:From vaccine candidate genes of L. interrogans serovar Lai strain #56601, eight genes were selected and analyzed by bioinformatic tools and proteomic strategies. These genes were cloned,expressed and purified by molecule clone methods and polyclonal antisera were gained by immunizing BABL/c mouse separately. Western blot and ELISA were used to identify the immunoreactivity of the OMPs with the rabbit serum of anti-L.interrogans strain Lai, respectively. The subcellar locations of these OMPs were analyzed by FACS, and the conservation of OMPs in fifteen pathogenic leptospires and an unpathogenic leptospires were identified by Western blot. In addition, we seleceted and identified the biological characters of LA3469 by bioinformatics, and examined the effects of iron on pathogenicity and growth of leptospira. And identified the protective immunity in vitro by adherence blocking test and inhibit Leptospiricidal activity test.Results: After analyzed the signal peptide, membrane structure and structural domain of these eight leptospira vaccine candidates, we found these vaccine candidates were all outer membrane proteins and had many T, B cell epitope which can evoke high immunological reaction. In our work, the prokaryotic expression system of the eight leptospira vaccine candidates were constructed by routine molecular biological techniques successfully and seven OMPs (LA0272, LA0301, LA0365, LA1404, LA1495, LA3469 and LB061) were expressed and purified, then polyclonal antibodies of these proteins were prepared.Then, identified the immunology characteristic of these vaccine candidates by fluoresence-activated cell sorter(FACS), Western blot and ELISA. These OMPs were found to react with serum of infected rabbit strongly and have high immunogenicity; The four genes (LA0301, LA1495, LA3469 and LB061) were deteced to encode outer membrane proteins; LA0301, LA1495 and LB061 are conserved in all Leptospires, LA1404 and LA1495 are only conserved in pathogenic leptospires. From these results there are four noval proteins have been found to be the potential efficacious leptospiral vaccines.In addition, LA3469 encodes an iron regulation protein, which annotated by COG analysis. From LA3468 to LA3471 may be an iron regulation domain of Leptospira sequence. The growth and pathogenicity of leptospira can be effected by iron. So the protein of LA3469 may be a pathogenicity protein. Then this protein could induce adherence blocking antibodies and inhibit Leptospiricidal activity. So LA3469 is an available vaccine candidate gene.Conclusions:(1) We selected the leptospiral vaccine candidates by reverse vaccinology, four novel genes (LA0301, LA1495, LA3469 and LB061) were screened to be potential efficacious leptospiral vaccine genes.We construct the technology basis for large screen the leptospiral vaccine genes.(2) In our work, we firstly analyzed the subcellar location of the seven proteins from leptospira by FACS to building a new way to identify the subcellar location of OMPs from leptospira.(3) Iron plays an important role in the growth and pathogenicity of leptospiras. The protein of LA3469 may be an iron regulating protein and a pathogenicity protein in infection. This protein can elicit protective immunity in vitro. So LA3469 is an available vaccine candidate.
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