Binding Of Peptide Binding Region Of Grass Carp MHC Class Ⅰ And Ⅱ Molecule To Invariant Chain

Major histocompatibility complex(MHC)is a group of closely linked and highly polymorphic transplanted genes involved in immune response,graft rejection and Immunomodulatory.It plays an important role in the body’s resistance to pathogen invasion and maintenance of its own stability.MHC-encoded molecules include MHC class I,MHC class Ⅱ and MHC class Ⅲ molecules.The MHC I molecule consists of a heterodimer consisting of a heavy chain(α)and a light chain(β2 microglobulin)disulfide bonds and is widely distributed in various parts of the body and expressed on all nucleated cell surfaces.MHC I plays a bridge role in the body’s immune response,recognizes and binds the endogenous antigen peptide,and then presents the endogenous antigen peptide to CD8~+T cells.The MHC Ⅱ molecule is a Type Ⅱ transmembrane glycoprotein,which also forms heterodimers by disulfide bonds between the alpha chain and the beta chain.MHC class Ⅱ molecules are more limited distribution,expressed only in antigen presenting cells,sperm and endothelial cells are also a small amount of expression.MHCⅡmolecules exogenous antigen peptide presented to the cell surface is recognized by CD4~+T cells to induce cellular immune response.The invariant chain(Ii)is a chaperone of the MHC Ⅱ molecule and plays an important role in the proper assembly,folding,maintenance of the structure of the MHC Ⅱ molecule and in assisting in the presentation of exogenous antigenic peptides.Because of its lack of polymorphism,it is named constant chain.It is found that the constant chain plays an important role in the participation of MHC molecules in immune response.However,in the past studies,it was only reported that Ii interacted with MHC molecules.However,it was not clear which Ii specifically interacted with which part of the MHC domains.Therefore,In this experiment,MHC class I and MHC class Ⅱ were divided into different functional domains,and the interaction between them was further studied by using prokaryotic expression system and eukaryotic expression system.Firstly,the sequences of grass carp MhcIα(Accession No:AB540144),MhcⅡβ(Accession No:JF436931)and invariant chain(Ii)(Accession No:KM369885)were obtained by logging on GenBank.Domain of the MHC gene.The cloned sequences were inserted into prokaryotic expression vectors pET-32a and pEGXT-4T-1 to get the corresponding recombinant plasmids.Escherichia coli Rosetta(DE3)is transferred into the recombinant plasmid,and the corresponding recombinant bacteria can be obtained by this method.After the recombinant bacteria were induced to express,the cells were harvested,repeatedly freeze-thawed and sonicated to obtain the corresponding protein.The GST tagged protein was purified by GST agarose gel The purified His-tagged protein was purified by Nickel ion chromatography.The purified protein was mixed and incubated and pulled down by nickel-ion chromatography.The interaction results were observed by SDS-PAGE gel electrophoresis and Western blot Verify the target protein.At the same time,the cloned genes were inserted into the eukaryotic expression vectors pEGFP-N1 and pmCherry-N1 to obtain the corresponding eukaryotic recombinant plasmids.The corresponding plasmids were co-transfected into 293T cells and observed in eukaryotic cells by laser confocal microscopy.Positioning and interaction with Ii.At the same time,by co-localization with organelles,the transport pathways of MHC molecules and Ii during antigen presentation were explored.The experimental results show that in the cell in vitro binding experiments,the peptide-binding region of the MHC molecule has the function of binding to Ii,and is not affected by the self-signal peptide,and losing the functional domain of the peptide-binding region does not have this function.In eukaryotic experiments,MHC molecules with a peptide binding region can also bind to Ii.At the same time,the MHC gene and the Ii gene expression product possessing the peptide binding region are localized on the inner and outer membranes of the cell,and the functional domain losing the peptide binding region loses its localization characteristic and is distributed throughout the cell.MHC molecules were expressed on various organelles(endoplasmic reticulum,Golgi apparatus,endosomes)and cell membranes,demonstrating all routes of delivery to antigenic peptides.