| Neospora caninum is an intracellular parasite with a wide host range.The surface proteins encoded by SRS family genes play an important role in mediating the adhesion and invasion of host cells.IL-18 can be produced by many kinds of tissue cells.IL-18 in conjunction with IL-12 can induce Th1 cells and NK cells to produce cytokines and cytotoxic activity respectively,promote the proliferation of T cell.Previous studies in our lab have found that the host can promote the clearance of Neosparo caninum by mediating the secretion of IL-18,inducing the secretion of IFN-γin serum,and promoting the Th1 immune response against Neosparo caninum infection.The aim of this study was to find a protein in the SRS family that had a better protective effect against the acute infection of Neosparo caninum,and to verify whether IL-18 as an adjuvant can enhance the protective effect of the recombinant protein.It could established a foundation for the development of Neosparo caninum vaccine.Construction and expression of recombinant plasmids of Neospora caninum SRSs,mouse IL-18 and NcSRS2-mIL-18.Three genes,SRS2,SRS17 and SRS52were selected by bioinformatics analysis.SRS2,SRS17 and SRS52 were amplified,and their sizes were 1041bp,1095bp and 921bp respectively.The results were tested in BLAST,and the nucleotide sequences of SRS2,SRS17 and SRS52 were 100%homologous with NCLIV033250（SRS2）,NCLIV068920（SRS17）and NCLIV069780（SRS52）recorded by Genbank.The mature peptide of mouse IL-18gene was amplified by using mouse cDNA（from primed mouse macrophage）as template,and its size was 471bp.The tandem recombinant vector pET-32a-NcSRS2-mIL-18 was successfully constructed with SRS2 and mIL-18 as templates.After the recombinant plasmid pET-32a-NcSRS2、pET-32a-NcSRS17、pET-32a-NcSRS52、pET-32a-mIL-18、pET-32a-NcSRS2-mIL-18 were transformed into BL21（DE3）,the molecular weight of the expressed protein were about 58kDa（SRS2）,60kDa（SRS17）,54kDa（SRS52）,37kDa（mIL-18）and 77kDa（SRS2-mIL-18）by SDS-PAGE analysis.These recombinant proteins could be expressed in the supernatant and the immunogenicities of these proteins were confirmed by western blot.In vitro blocking of polyclonal antibody against recombinant proteins of Neospora caninum tachyzoites.The serum of mice immunized with recombinant protein was diluted and incubated with the tachyzoites of Neospora caninum.The effect of antiserum on the invasion and proliferation of Neospora caninum was observed.The results showed that the polyclonal antibodies against SRS2,SRS17 and SRS52 could inhibit the adhesion and invasion of Neospora caninum to host cells.Immunoprotection of recombinant proteins SRSs,mIL-18,SRS2-mIL-18against Neospora caninum infection in mice.BALB/c mice were randomly divided into seven groups,including five experimental groups（SRS2,SRS17,SRS52,mIL-18and SRS2-mIL-18）,negative and positive control groups.The experimental groups were immunized subcutaneously for three times respectively,and the mice were inoculated intraperitoneally with Neospora caninum tachyzoites 15 days after the three immunizations,and the survival and weight changes of the mice were observed and recorded every day.The sera of mice were collected 10 days after the three immunizations to determine antibody and cytokine production levels by ELISA.After20 days,the tissues of heart,liver,spleen,lung,kidney and brain were collected to make pathological sections and the pathological changes were observed.The results showed that the IFN-γsecretion level of the mice immunized with SRS2-mIL-18 was significantly higher than that of the mice immunized with SRS2（P<0.05）.In addition,IFN-γin SRS17 group was higher than that in SRS2 and SRS52 groups.Compared with the positive control group,the survival rate of mice in each group was16%in the positive control group,67%in the SRS52 group,83%in the SRS2 and mIL-18 groups,and 100%in the SRS17 group and the SRS2-mIL-18 group as well as the negative control group.There was no obvious lesion in group SRS2-mIL-18,and the number of parasites in brain tissue was the significantly decreased.Compared with NcSRS2,NcSRS17 and NcSRS52 proteins can induce humoral and cellular immune responses,which can provide protections for mice,and the protective effect of SRS17protein was better.Construction and expression of recombinant plasmids of goat IL-18 and NcSRS17-gIL-18.The IL-18 gene of goat（gIL-18）was successfully amplified,the size of which was 471bp.The results were compared with BLAST,and 100%homology was found between the gene and NM001285544.1 of goat in Genbank.The tandem recombinant vector pET-32a-NcSRS17-gIL-18 was successfully constructed with SRS17 and gIL-18 as templates.After BL21（DE3）was transformed,the protein molecular weight was about 37kDa and 73kDa by SDS-PAGE analysis.The recombinant protein could be expressed in the supernatant,and its immunogenicity was confirmed by western blot.Immunoprotection of recombinant proteins SRS17 and SRS17-gIL-18against Neospora caninum infection in goats.Eight goats were randomly divided into four groups,including two experimental groups（SRS17,SRS17-gIL-18）and two control groups（positive control and negative control）.The goats were immunized with recombinant proteins SRS17 and SRS17-gIL-18.The antibody titer and cytokine level of goat serum were detected after immunization.15 days after the last immunization,1×109 Neosparo caninum were inoculated into the blood of each goats in the experimental group and the positive control group.The goats were humanely euthanized under anesthetization 40 days after the infection.The heart,liver,spleen,lung,kidney and brain tissues were sectioned and the pathological changes were observed after HE staining.The brain genome of each group was extracted and detected by fluorescence quantification PCR.The results showed that the serum titer of SRS17 group was about 1×105,and that of SRS17-gIL-18 group was about 2×105.And the levels of IL-4,IL-12 and IFN-γcytokines in each experimental groups were increased significantly compared with the negative group,indicating that the recombinant proteins of SRS17 and SRS17-gIL-18 can stimulate the immune response of Th1/Th2.In addition,the level of cytokine secretion in SRS17-gIL-18group was higher than that in SRS17 group,indicating that IL-18 improved the protective effect of SRS17.The results showed that compared with the positive control group,the pathological changes of each tissue in the two experimental groups were not obvious.The results of the fluorescence quantification PCR showed that SRS17 and SRS17-gIL-18 could significantly reduce the amount of parasite in goat brain compared with the positive control group. |
