Preliminary Crystallographic Study Of The Superantigens SEN And SEQ

Superantigens(SAgs)are a special class of antigen molecules that can cause nonspecific activation of T cells,which could result in a strong immune response,triggering serious clinical symptoms such as toxic shock syndrome,multiple organ failure and autoimmune diseases accordingly.On the other hand,superantigen can stimulate the body to produce a large number of biological active cytokines and enhance the body’s killing activity against tumors.Staphylococcus entertoxin N(SEN)and staphylococcus entertoxin Q(SEQ)two novel superantigens identified in recent years have been found to play an important role in numerous food poisoning episodes.To date,the mechanism by which SEN and SEQ trigger immune effects have not yet been clarified.In this study,sen and seq gene were obtained by gene amplification method.The sen and seq gene were cloned into the prokaryotic expression vector pGEX-6P-1 to obtain recombinant expression plasmids pGEX-6P-1-sen and pGEX-6P-1-seq,respectively.The constructs were introduced into E.coli BL21(DE3)strains to obtain SEN and SEQ expression strains,respectively.Recombinant SEN and SEQ proteins were overexpressed in Escherichia coli.The E coli cells were disrupted to obtain soluble proteins.A series of chromatography methods such as GST affinity chromatography,Mono Q ion exchange chromatography,and Superdex 200 size exclusion chromatography were used to obtain high-purity SEN and SEQ proteins.Crystallization kits were used to crystallize the SEN and SEQ proteins by sitting-drop vapour-diffusion method.Finally,high-quality crystals of SEN and SEQ proteins were obtained under 2%(v/v)Tacsimate ~TMM pH 4.0,0.1 M Sodium acetate trihydrate pH 4.5,12%(w/v)polyethylene glycol 3350 and 6%(v/v)Ethylene glycol,0.1 M Citric acid pH3.5,8%(w/v)polyethylene glycol 6000,respectively.The crystals were verified as protein crystals by SDS-PAGE and Western blot.X-ray diffraction datas of SEN and SEQ crystals were collected at 2.0?and 3.0?resolution,respectively.Finally,SEN and SEQ protein structures were successfully resolved by data processing software such as HKL-2000、iMosflm、CCP4、COOT、Phenix.This study laid the foundation for the fine structure analysis of SEN,SEQ and SEN/SEQ-MHC-TCR ternary complex,which helps to elucidate the mechanism of action of SEN and SEQ from a structural perspective,thus providing a theoretical basis for drug design and disease prevention.