The Development Of Superantigen SEB Vaccine RS-H And The Study Of Its Immune Effect

Staphylococcus aureus plays an important role in human food poisoning, soft tissue, and bone infections, as well as potentially lethal toxic shock. This common bacterium synthesizes various virulence factors that include staphylococcal enterotoxins(SEs). Picomolar concentrations of Superantigens are able to bind directly to major histocompatibility complex class II on antigen-presenting cells and specific Vβ regions of T-cell receptors, resulting in rapid hyper-activation of the host immune system,and then ultimately eliciting various proinflammatory cytokines that can induce fever, acute lung injury, Acute respiratory distress syndrome,multi-organ failure, and lethal shock.Treatment of Superantigen have received attention in the world,many therapy against SEs based on a variety of antibodies,vaccines and other small molecules have begun.In this study,By using molecular cloning method to fuse the SEB gene which lost TCR Vβ binding ability and Mycobacterium tuberculosis heat shock protein 65(HSP65) gene,constucting the recombinant plasmid pET-28a-rS-H. We succesfully obtained a noval SEB vaccine-recombinant vaccine rS-H under induction of IPTG.After optimizing the expressiong and purification process,we conclude that: By optimizing the inducing expressiong conditions,the amount of protein rS-H increased by about 15%; By optimizing the washing conditions,cracking conditions as well as the refolding conditions for the inclusion body rS-H,the amount of target protein rS-H in the crude purification productions raised by about 40%;By optimizing the sample flowing through a metal chelate chromatography and the gradient elution length of anion exchange chromatography on Q,the purity of the recombinant protein can be increased to more than 90%, the final yield can be increased to over 45%.By studying of the immune effect of the vaccine rS-H,we conclude that: After immunizing subcutaneously three times, by analysising the SEB specific antibody titer result of each group,protection rates of each group after toxin challenge experiments as well as the neutralizing ability of antibody in vitro,the recombinant vaccine SEB induced humoral immune response successfully, the highest value of the anti-SEB specific antibody titer was close to 215, significantly higher than other immune group(P <0.01); After challenge of 5×LD50 does and 2×LD50 does of SEB,the protection rate were 80%(i.n.) and 90%(i.p.) respectivly,significantly higher than other immune group(P <0.05); And effectively relieve,the lungs, hearts and kidneys tissues damage caused by bleeding and lesions after challenge; After challenge of LD50 and LD10 doses of toxin, the rS-H vaccine can inhibit various cytokines increase,significantly lower than the negative control group(P <0.01);In vitro challenge experiments, rS-H vaccine-induced antibodies can effectively neutralize SEB and can inhibit various inflammatory cytokines induced by SEB,which was significantly lower than the control group(P <0.05). These results show that the purified rS-H can induce a humoral immune response effectively and induced anti-SEB antibody successfully,and also relieve symptoms caused by SEB effectively.