Study On Biological Functions Of Several Novel Recombinant Staphylococcal Enterotoxins

Staphylococcal Enterotoxins (SEs) are soluble extracellular proteins excreted by plasma-coagulase or thermonuclease positive staphylococcus bacterial strains, sharing smilar structures and virulence, and included in superantigen family. Besides acute gastro-enteritis,Kawasaki disease,dermatosis and respiratory diseases are associated with SEs. Up to date, over 20 types of ses and ses-like genes were discovered, but the research about novel SEs were only remained on gene detection level. The purpose of this study was to reveal the toxicity and superantigen activity of several novel SEs.Recombinant SEA-HIS\ SEO-HIS\ SEG-HIS\ SEK-HIS\ SEU-HIS\ SEQ-HIS were expressed by 6 pET28α-ses vectors in E.coli.BL21 (DE3) and purified by nickel chelate affinity chromatography method. Recombinant proteins were injected into 4-week-old mice, at the doses of 20μg/g or 5μg/kg, via tail vein, PBS was used as a blank control. 72h later, mice were sacrificed after taking blood sample by extirpating eyeballs and thymuses/spleens/livers were obtained. Viscera indexes, activity of heterologous compound metabolic enzyme, transcriptions and expressions of cytokines (IL-2\ IL-4 IL-6\ TNF-α\ IFN-γ) were evaluated in vitro. Cell proliferations, apoptosis, transcriptions and expressions of cytokines (IL-2\ IL-4\ IL-6\ TNF-α\ IFN-γ) were evaluated in vivo.At the dose of 20ug/g, the transcription of IL-2\ IFN-γinvolved in humoral immunity and IL-4\IL-6 engaged in cell-mediated immunity were suppressed by SEA-HIS and SEQ-HIS. The transcription and expression of IFN-γinvolved in inflammatory response were promoted by SEK-HIS\SEG-HIS and SEO-HIS. The activities of POD\ SOD\ GSH-PX\ LDH in the liver were affected by SEA\ SEO\ SEG\SEK\ SEQ. At the dose of 50ug/mL, the transcription of TNF-αwere promoted by SEA\ SEO\ SEG\SEK\ SEQ.The effects of novel SEs-HIS (except SEU-HIS) on cytoproliferation were smilar to the effect of classical Staphylococcal enterotoxin A, suggesting that novel SEs-HIS (SEO-HIS\SEG-HIS\SEK-HIS\SEQ-HIS) were superantigens. Furthormore, MHCII and diverse types of Vβin TCR were required in cytoproliferation. Sequence of the superantigen activities from the highest to the lowest is below: SEK-HIS\ SEQ-HIS\ SEA-HIS\ SEG-HIS\ SEO-HIS, SEU-HIS didn't beong to superantigen family. This result was the same as the predictions according toα3-β8 loop and Zn2+ sites in the structure of proteins. The transcriptions and expressions of IL-2\ IL-4\ IL-6\ TNF-α\ IFN-γwere promoted at the dose of 5μg/kg in vivo studies and 4μg/mL in vitro studies.To sum up, SEA\ SEO\ SEG\SEK\ SEQ were regards as toxic substance because of affecting activities of exogenous compound metabolism Enzymes in the livers and introducing inflammatory response. SEA\ SEO\ SEG\SEK\ SEQ were regards as super antigens because of affecting the transcriptions and expressions of IL-2\ IL-4\ IL-6\ TNF-α\ IFN-γand promoting lymphocyte proliferation. New problems of Food Safety were interrogated due to toxicity and super antigen acitivity of new Staphylococcal Enterotoxins.