Study On The Interaction Between Superantigen And Human TCR-CD3 Complex

Superantigens cross-link MHC class ? and TCR on antigen presenting cells and activate most T cell populations,which can cause a large proportion of immune responses.Superantigens cause a large number of T cell proliferation by simultaneously binding MHC ? and T cell receptors on T cells.We need to further explore the interaction between superantigens and T cells,which will help us to study the mechanism of lymphocyte immune tolerance to self antigens.In this paper,the expression and purification of superantigens SEA and SEH,human-derived ??TCR/CD3 complex and p MHC protein were expressed and purified.Then combine with each other to study the interaction of superantigen with human-derived ??TCR/CD3 complex and p MHC.First,the gene fragments of superantigens SEA and SEH were inserted into p ET-21a(+)plasmids,respectively,and expressed in E.coli.The ? and ? subunits of the same ??TCR were inserted into the same p EB6 plasmid.The delta,epsilon,gamma,and zeta subunits of CD3 were inserted into the same pc DNA3.1 plasmid.They are expressed in mammalian cells.The p MHC ? gene fragment was inserted into the p Fast Bac Dual plasmid so that it was expressed in insect cells.Enrich the protein by affinity chromatography.Separate the protein by molecular sieve.The purified protein was used for interaction studies.First,we explored the interaction between superantigen and ??TCR/CD3 complex.Then the interaction between superantigen,??TCR/CD3 complex and p MHC protein was investigated.Finally,we explored the effects of crosslinker and Zn2+ concentration on the interaction of superantigen,??TCR/CD3 complex and p MHC.The experimental results found that the superantigens SEA and SEH are ideally expressed in the prokaryotic expression system.The expression of human-derived ??TCR/CD3 complex in mammalian cells is low,making it difficult to obtain large amounts of protein.After optimizing the conditions,p MHC can obtain enough protein in insect cells.In the interaction study of superantigen with ??TCR/CD3 and corresponding p MHC,it was found that SEH has a good combination with 2IAL TCR/CD3 and p MHC,which can be used for transmission electron microscopy observation.The cross-linking agent can increase the yield of the formed complex but may cause the morphology of the complex to change and affect the analysis of the experimental results.