Expression, Analysis On Structures & Bioactivities Of Staphylococcal Enterotoxin G And Enterotoxin U

Staphylococcal enteortoxins( SEs) is a exotoxin produced by Staphylococcal aureus, which is one of the most important pathogenic factors of staphylococcal food poisoning. More and more enterotoxin genetypes were detected from different staphylococcus isolates, while lacking of verification results based on those new enterotoxins and their functions. It is meaningful to obtain those staphylococcal enteortoxins and to analysis their structures and functions. In paper, the relationships between the structures and biological function of staphylococcal enteortoxins were analysed by purified SEG, SEU.A highly conserved seg gene and selu gene with a samesense mutation by comparing with other reported sequences in GenBank were successfully cloned. The prokaryotic expression systems of seg gene and selu gene were constructed. The soluble recombinant proteins GST-SEG, GST-SEU were purified by a single step of affinity chromatography using Glutathione Sepharose 4B column. GST-SEG was approximately 24% of the total bacterial proteins, and GST-SEU was 22%. SEG, SEU proteins were purified from GST-SEG, GST-SEU by removing GST.The biological activities of SEG and SEU were evaluated. MTT colorimetric was applied to examine the effect of enterotoxins at different dosages on promoting proliferation of ICR mouse spleen lymphocytes. The cytotoxicity of enterotoxins was measured by examining the activity of Caspase-3 and analyzing the apoptosis of DNA ladder antioxidant activities, while the changs of oxidase-activity caused by enterotoxins was also examined in vivo. The results showed that low dosage of enterotoxins promoted the proliferation of ICR mouse spleen lymphocytes, higher dosages lead to apoptosis and enterotoxins could reduce the activities of oxidases. SEU displayed lower biological activity than SEG.The secondary structures of SEG, SEU protein were plotted in terms of the results of CD spectroscopies and secondary structure predications by bioinformatics analysis. The amino acid amounts ofα-helical,β-sheets,β-turn and random coil of SEG protein were respectively 35, 89, 35 and 106, while the amino acid amounts ofα-helical,β-sheets,β-turn and random coil of SEU protein were approximately 33, 71, 22 and 106. The three-dimensional structure of SEU was constructed by homology modeling method. The molecular basis of biological activities were analysed by comparing the amino acid sequences, secondary structure and three-dimensional structure of SEU with SEB and SEC3. Lys56, Asn60, Ala87 and amino acids located in 211-215 of SEU were predicted as the function sites. SEU possessed potential binding sites of TCR Vβ, and lower binding activity to MHCⅡmolecular than SEG.