| Cytokines are multifunctional soluble proteins or glycoproteins that had high activity produced by immune cells and in many cases of other cell types.They act as chemical communicators among cells and are regulators of host defence against pathogens and the inflammatory response.Interleukin-2,interleukin-4,interleukin-18 and interferon-gamma are all necessary for immune response and become a hot research in immunoadjuvant because of their regulating function.This research aimed at certifying the immunological adjuvant effect of recombinant IL-2,IL-4 and IFN-y of porcine on the foot-and-mouth disease synthetic peptide vaccine and testing the bioactivity of the recombinant protein IL-2,IL-4,1L-18 and IFN-γ of goat and so does the restraint bioactivity detection of antiserum to corresponding protein.1.Prokaryotic expression of porcine IL-2,IL-4 and IFN-y gene and bioactivity detection of recombinant proteinRosetta2 with the gene encoding IL-2,IL-4 and IFN-γ of porcine respectively were used to express the recombinant protein induced by IPTG.The recombinant proteins were approximately 21.4 kDa,30.3 kDa and 20.7 kDa respectively and mainly existed in inclusion bodies.Through improved purification and renaturation,highly purified recombinant proteins were obtained.The bioactivity test proved that purified recombinant protein IL-2 could significantly stimulate the proliferation of porcine peripheral blood lymphocyte in a dose-dependent manner and the density of 10 μg/mL presented the highest stimufation.But the recombinant protein IL-4 couldn’t stimulate the proliferation of lymphocyte effectively.The activity of purified porcine IFN-γ protein against VSV was about 1.135×104 U/mg.2.Immunological enhancement of recombinant porcine IL-2,IL-4 and IFN-γ on foot-and-mouth disease synthetic peptide vaccineThe recombinant protein IL-2,IL-4 and IFN-γ were injected with the porcines at the concentration of 15 μg/kg companied by the foot-and-mouth disease synthetic peptide vaccine respectively.And the PBS control group,vaccine control group and 28a/32a protein control group were set at the same time.The changes of antibody and IL-4,IL-10,IL-17 and IFN-y levels of porcines were tested by ELISA.The results showed that the recombinant protein we made all had good biological activity.IL-2 and IFN-γ protein significantly increased the level of antibody against foot-and-mouth disease(P<0.01),while IL-4 appeared to have a suppressive effect(P<0.01).Recombinant IL-4 significantly enhanced IL-4 and IL-10 secretions compared with that of the other groups(P<0.05).The IL-2 and IFN-γ could significantly stimulate the production of IFN-γ(P<0.01).These results indicated that IL-2 and IFN-γ of porcine could enhance the immune response against FMDV significantly.3.Prokaryotic expression of goat IL-2,IL-4,IL-18 and IFN-γ gene and the restraint bioactivity detection of antiserum to recombinant proteinEscherichia coli BL21 with the gene encoding IL-2,-IL-4,IL-18 and IFN-y of goat respectively were used to express the recombinant protein induced by IPTG.The recombinant proteins were approximately 35.6 kDa,30.7 kDa,22.3 kDa and 37.3 kDa respectively and mainly existed in inclusion bodies.After purification and renaturation,highly purified recombinant proteins were obtained.The bioactivity test showed that the recombinant protein IL-2,IL-4 and IL-18 were able to stimulate the proliferation of goat peripheral blood lymphocyte in a dose-dependent manner,and the concentration of 5μg/mL in IL-2,25 μg/mL in IL-4,4 μg/mL in IL-18 presented the highest stimulation.The recombinant protein IL-18 could significantly promote the phagocytosis neutral red of peritoneal macrophages at the concentration of 0.25 μg/mL,and increase NO in culture supernatants of peritoneal macrophages at the concentration of 1μg/mL.The activity of purified goat IFN-y protein against VSV was about 2.52×104 U/mg.The antiserum of corresponding recombinant protein could restrain the bioactivity of protein. |
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