Characteristic Of Binding Of Ii To MHC Molecules In Grass Carp(Ctenopharyngodon Idellus)

The major histocompatibility complex(MHC)is a group of closely linked genes with polymorphisms that play an important role in vertebrate transplant rejection,immune response,and immune regulation.MHC is mainly divided into MHC class Ⅰ and MHC class Ⅱ molecules.Invariant chain(Ii)is a chaperone protein of MHC class Ⅱ molecules and belongs to type Ⅱ transmembrane glycoprotein.Studies have shown that Ii can bind to MHC class Ⅱ molecules and assist in the presentation of exogenous antigenic peptides.In this process,Class Ⅱ-associated invariant chain(CLIP)plays a key role.Ii can also bind to MHC class Ⅰ molecules and participate in endogenous antigenic peptides.To date,there have been few reports on the binding characteristics of Ii and CLIP to MHC molecules in fish.In order to understand the relationship between grass carp Ii/CLIP and MHC molecules in the process of binding,and further explore the role of Ii in the antigen presentation process,this paper mainly carried out the following aspects.First,the expression and purification of recombinant plasmids and proteins were constructed.Primers were designed based on the Mhc Ⅰα,Mhc Ⅰβ,Mhc Ⅱα,Mhc Ⅱβ and Ii gene sequences of grass carp(Ctenopharyngodon idellus)in GenBank,and the laboratory-preserved the gene of Mhc Ⅰα,Mhc Ⅰβ,Mhc Ⅱα,Mhc Ⅱβ and Ii was used as a template,and the corresponding gene of interest was cloned and purified,and inserted into prokaryotic expression vectors pET-28a,pET-3 2a and pGEX-4T-1 to construct corresponding recombinant plasmids.The recombinant plasmid was then transformed into E.coli Rosetta(DE3)to obtain the corresponding recombinant strain.The expression of the corresponding protein was induced by isopropy 1-β-D-thiogalactoside(IPTG).The results showed that these target genes could correctly express the corresponding protein of interest.Purification of the protein was carried out according to the tag of the carrier,and the SDS-PAGE gel electrophoresis showed that the pure protein of interest was obtained.Secondly,studies were conducted on the binding characteristics between MHC and Ii and CLIP molecules.The binding activity of the related proteins was detected by Pull-down technique.The results of Western blot showed that Ⅰα,Ⅱα and Ⅱβ of MHC molecules of grass carp could bind to Ii or CLIP in vitro without binding to Ip.The results of competitive binding of MHC molecules to CLIP and Ii showed Ii and CLIP can combine Ⅰα,Ⅱα and Ⅱβ of MHC molecules without binding to Ⅰβ.The results of competitive Pull-down test of MHC molecule and Ii or CLIP show that both CLIP and Ii can form a trimer structure with MHC molecules.Finally,the localization and binding of MHC molecules to Ii and CLIP were studied intracellularly.The cloned gene was inserted into the eukaryotic expression vector pEGFP-N1 and pmCherry-N1,and the corresponding eukaryotic recombinant plasmid was obtained and co-transfeeted into 293T cells.The molecules in eukaryotic cells were observed by laser confocal microscopy.The colocalization between the two results showed that Ii can colocalize with the four strands of MHC molecules in the cell,while CLIP can only partially localize with MHC molecules.Colocalization of recombinant plasmids and organelles shows that Ii can interact with cell membrane,endosome,endoplasmic reticulum colocalization with the Golgi apparatus,and CLIP can only be localized with the Golgi apparatus.Co-immunoprecipitation confirmed the intracellular interaction between Ii or CLIP and MHC molecules.The results showed that both Ii and CLIP can co-precipitate with MHC molecules.The results of this study have identified the organelles(cell membrane,endocytosis and endoplasmic reticulum)in the eukaryotic cells of MHC Ⅰα,Ⅰβ,MHC Ⅱα,Ⅱβ,Ii and CLIP proteins.Grass carp Ii can bind to MHC class Ⅰ molecules(Ⅰα and Ⅰα),respectively.And four chains of class Ⅱ molecules(Ⅱα and Ⅱβ),which form two trimers(Ⅰα/Ⅰβ/Ii andⅡα/Ⅱβ/Ii),while CLIP of Ii binds to MHC Molecules form a key active fragment of the trimer.These results enrich the theoretical knowledge of the relationship between fish Ii and MHC molecules,providing an experimental basis for further research and exploration of the role of Ii in immune response.