| Interleukin-18(interleukin-18, IL-18) is a kind of Interleukin, it can induced T cells to producte a large number of IFN-y, so was previously called IFN-y inducing factor. IL-18has potential applications in enhancing immunity,anti-tumor,anti-pathogenic microbial infection,inhibit viral activity. Interferon gamma (IFN-y) is also known as a kind of immune interferon, which plays a role in inhibiting viral replication,antitumor, antiparasitic activity and immune regulation. In this study, porcine IL-18(pIL-18) and porcine IFN-y (pIFN-y) gene were expressed in Lactococcus lactis.Then the pIL-18, pIFN-y protein was used with Pseudorabies Vaccine in order to research the effect about the immune response which pIL-18and pIFN-y played on.To extracted total RNA from pig spleen lymphocytes, According to porcine interleukin-18and porcine Interferon-y, pair of primers were designed. pIL-18and pIFN-y were amplified by RT-PCR. The pIL-18and pIFN-y gene were inserted into plasmid pProExHTa to construct recombinant plasmid pProExHTa-pTL-18, pProExHTa-pIFN-y, and transformed into E. coli. SDS-PAGE showed that the recombinant protein were successfully expressed, and mainly in the form of inclusion bodies. Mice was injected intramuscularly with purified recombinant protein to obtaining pIL-18, pIFN-y antiserum.The serum titer is1:25600.Secreted plasmid pAMJ399induced by Lactococcus lactis pH is used as expression vector, to construct the recombinant plasmid pAMJ399-pIL-18, pAMJ399-pIFN-y, and then pAMJ399-pIL-18, pAMJ399-pIFN-y is transformed to Lactococcus lactis MG1363by electricity facing, to construct recombinant Lactococcus lactis pAMJ399-pIL-18/MG1363, pAMJ399-pIFN-y/MG1363, which induced by the acid production. SDS-PAGE and Western blot results show that there were about19.25ku and17.71ku protein in the culture supernatant and the cells, which have specific reaction with pIL-18, pIFN-y antiserum from mice. So pIL-18, pIFN-y protein is expressed in recombinant Lactococcus lactis, which can exist in medium in the form of secretion and in cells.For the detection of antiviral activity of pIL-18, pIFN-y expressed in recombinant Lactococcus lactis, supernatant of recombinant strains is filtered and incubated with Vero cells for24h, Cytopathic effect can be observed by the microscope when cells is inoculated swine pseudorabies virus (PrV) and Porcine Epidemic Diarrhea Virus (PEDV) for60h. The results showed that cells treated with pIL-18, pIFN-y were good, cells of control without pIL-18, pIFN-y are wrinkled, agminated, round, deciduous. The statistical analysis results by MTT showed that supernatant group with pIL-18, pIFN-y compared with virus group have significant difference extremely (P<0.01). pIL-18, pIFN-y of supernatant of recombinant Lactococcus lactis have antiviral activity.In order to explore the effects of this immunoadjuvant, after the recombinant strains was induced, we concentrated the cultured supernatant, and pIL-18,pIFN-y,pIL-18with pIFN-y were used to combine with Pseudorabies Vaccine. The groups were boosting injected two weeks later. The anti-PrV immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice at7、14、21and28days after first immunization. The results of ELISA showed that the groups of vaccine combining with pIL-18, pIFN-y had higher antibody level than the group of vaccine. And in the3groups with different protein, the antibody level of pIL-18with pIFN-y group was higher than other2groups, and had significant difference (p<0.01) compared with the vaccine group. The pIL-18groups indicated significantly difference (p<0.05) than vaccine group. Meanwhile, we detected the antibodies’ neutralization titer by cell neutralization test, the neutralization titer of vaccine group was1:45.26, a vaccine combining with pAMJ399/MG1363group was1:44.74, vaccine combined with pIL-18alone group was1:62.6, vaccines combined with pIFN-y group was1:53.4, vaccine combined with pIL-18and pIFN-y was1:66.36. The spleen lymphocyte proliferation activity of immunized mice was tested by MTT assay. The result showed that lymphocytes proliferation index of vaccine combining with pIL-18、pIFN-γ group was significantly difference (p<0.01) compared with vaccine group and the group of vaccine combining with pAMJ399/MG1363. It indicated that vaccine combining with pIL-18-pIFN-y group induced the body producing a higher level of specificity immune response.14days after boost immunity, all experimental mice were challenged with PrV then observed the morbidity and deaths of each group. The results showed that the protective rate of vaccine group and vaccine combining with pAMJ399/MG1363was83%and the group of vaccine combining with pIL-18and pIFN-y was100%. All the results showed that IL-18、 IFN-y had good immunological effect as adjuvant.In summary, the results obtained in this study showed that pIL-18、pIFN-γ protein expressed in Lactococcus lactis had good immunogenicity and biological activity, and when used it with vaccine, it showed good adjuvant features, this provides experimental data and theoretical basis for the application of the pIL-18,pIFN-y, and laid the foundation for the further study of other vaccine adjuvants. |
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