| The family of staphylococcal enterotoxins (SEs), including SEA-E, SEG,SEH and SEI, has been known to contribute to a broad spectrum of diseases ranging from tissue infections to life-threating speticemia and toxic shock syndromes, and from Kawasaki's syndrome to atopic dermatitis, even multisystem vasculitis. Moreover, SEs are also manufactured as bio-logical warfare agents and used on military actions. SEA, SEB, SEC and SED are common SEs and possess the high toxicity among SE family. In recent years, much attention has been paid to the study on immunizing treatment of the diseases caused by SEs. Inhibitory peptides and mutants were designed and proved to be effective in antagonising SEs. Because the SEs are comprised of so many toxins, development of the broad-spectrum inhibitory peptides may be a promising method for antagonising SEs. Moreover, more researches were concerned about the SEA, SEB and SEC, so few studis on fighting with SED were carried out. This study is designed to explore the above issues. The main content and results of this study are as follows:Firstly, nine low-molecular peptides were designed on the basis of high conservative re-gions of amino acid sequences and structures of the SEs. All peptides with more than 95% purity were constructed by solid-phase synthesis and can be used in the subsequent study. Secondly, we constructed the human PBMC cellular models. Lymphocyte proliferation assay and detection of IL-2, IFN-γand TNF-βby ELISA were used to screen the inhibi-tory peptides. The results indicated peptide P72 possessed a broad-spectrum antagonist activ-ity against superantigen SEA, SEB and SEC. However, the other eight peptides had no inhib-iting activity. The viability studies indicated that the PBMCs remained similar livability, with or without the addition of peptide P72, and the peptide P72 didn't influence the activation of PHA and ConA stimulating proliferation of PBMCs.Thirdly, the"two-hit"animal model with combination of LPS and the SEs was carried out in the murine experiments. The minimal doses of superantigen that had been shown to cause 100% lethality were obtained. Peptide P72 on the biological activity of SEs was de-tected by this animal model, the results showed P72 can completely protect most of the mice for each toxin tested.Fourthly, we detected the ability of the peptide P72 binding to MHC classⅡmolecules by competition assay and found that the P72 could not bind to MHC classⅡmolecules, which indicated that the inhibitory activity of P72 may not due to binding to MHC classⅡmolecules.Fifthly, we compared the amino acid sequences between P72 and SEs, and constructed the three-dimension structure of P72. The results showed three-dimensional domains of the peptide P72 from SEA, SEB and SEC were quite similar.Finally, a SED mutant was constructed and named SEDN23A/H26R. We detected the mito-gen activity of SEDN23A/H26R and found that its mitogen activity decreased significantly. Fur-thermore, competition assay was used to detect the ability of mutant SEDN23A/H26R binding to MHCⅡ. The TCRVβspecificity of the mutant was then determined with FACS. The results showed that residue N23 played an important role on SED interacting with human TCRVβ5. Residue H26 was probably an active site of SED binding to other human TCRVβ.In conclusion, our results indicate that P72 possesses a broad-spectrum antagonist activ-ity against superantigen SEA, SEB and SEC. Inhibitory activity of P72 may not due to bind-ing to MHC classⅡm olecules. N23 and H26 on SED are the important residues involved SED interacting with TCRVβ.SEDN23A/H26R with lower mitogen activity may be used to manufacture the attenuated vaccine. Our study might enrich understanding the mechanism of immune response arised by superantigens, and provide new methods for developing counter-measure to fight with SEs-related deseases and biological weapons.
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