Studies On Staphylococcus Aureus Enterotoxin B As Adjuvant

The frequency distribution of animal diseases caused trouble to breeding industry, par-ticularly the viral diseases.Because there's no effective treatment, an immune prevention is essential. Currently there are various types of livestock and poultry vaccines which bo-th have advantages and disadvantages.The traditional vaccines are out gradually because of low prevention and insecurity; novel vaccines which with high security are researched, such as subunit vaccines, recombinant vaccines and synthetic peptide vaccines, but these vaccines are weak of immunognicity, the ordinary adjuvant can not realize the needs.The development of novel adjuvant with high activity and low side effects are more urgent.The adjuvant can enhance the body's immune response against the antigen, it is usually used at the same time or before of the antigen adminitration. Many adjuvants were stud-ied, and their main function is enhancing immune response, reducing the antigen dosage and immunization times, accelerating the effective immunity of the poor immune respo-nse groups, accelerating the speed of the immune response and prolonging the duration of immunity and so on. Such substances with this immune function are potentially adju-vants.Staphylococcus aureus enterotoxin B (SEB) is a kind of exotoxin superantigen prope-rties with MHC non-restricted. It can activate peripheral blood T cells, regulate NK cells, stimulate lymphocytes to produce TFN-a and activate Thl cells.SEB can stimulate T cell to improve immunity, especially cellular immunity.SEB has potentiality to be an adjuvant because it can activate many immune cells and cause lots of cytokines release.So following researches were explored:According to the published DNA sequence of SEB in GenBank, a pair of specific pri-mers were designed and synthesized. SEB gene was amplified from DNA of Staphyloco-ccus aureus Genome by PCR and cloned into the pMD18-T vector. The recombinant pla-smid was identified by restriction-enzyme analysis and sequencing. The sequence BLA-ST.showed SEB gene has 100% homology with PM36 strain. The prokaryotic expression plasmids (pKG-SEB and pET-28a-SEB) were construced and expressed in E.coli BL21 (DE3)successfully. The expressed pET-28a-SEB fusion protein was about 28 KD,and the expressed pKG-SEB fusion protein was 54KD.The prokaryotic expression plasmid(pKG-HA1)was transformed into E.coli BL21 (DE3)the positive clones were expressed. The expressed fusion protein in E.coli BL21 (DE3) was in supernatant and inclusion body by the analysis of SDS-PAGE and Western- blotting. The fusion protein is about 60 KD as expected and most of protein existed in inclusion body which was very useful for animal Experiment.Two weeks old ai wei yin broilers were immunized with AIV H9/NDV vaccine and different concentration of recombinant SEB,AIVH9/NDV combined vaccines and diff-erent concentration of standard SEB,respectively. Both recombinant and standard SEB can increase the immune response by testing the antibody titers and lymphocyte activity. The results showed that recombinant SEB had better immune enhancement at the dosage of 5μg; In the early stage of immunity, the antibody titer was increased rap-idly in SEB immunized groups compared with control group;in the first and second weeks after im-munization, spread about 2 antibody titer, to the fourth week, the antibody titer in SEB and vaccine combined immunized group was still higher than that in the control group; lymphocyte activity was highest in the 15th day after immunization, the value in the im-munized group with the SEB vaccine was higher than that in the control group.Two weeks after immunization ai wei yin broilers with 5μg of recombinant SEB and AIV H5 inactivated vaccine,5μg of recombinant SEB and HA1 protein, respectively. The results showed that recombinant SEB could enhance immune response to the inac-tivated vaccine and the subunit vaccine.SEB has the ability of immune activation, which was confirmed by three animal exp-eriments independently. All these data showed that SEB can improve humoral or cellular immune response.