Characters Of The Location In Cells And Binding Activity Of Duck Mhc And Ii Gene Expressed Products

Major histocompatibility complex(MHC)is a highly polymorphic protein encoded by a group of genes located in the region of the vertebrate specific chromosome region.MHC molecular distribution in different cell surface involved in antigen presentation and immune response.Different kinds of animal MHC and its encoding product names are different.The ? class MHC molecule family is ISO two dimers composed of alpha and beta two chains.It is with the gamma chain polymerization in the endoplasmic reticulum,the formation of nine dimers.The gamma chain in the dimer is different from the alpha and beta chains,and is non polymorphic,so it is called the chain(Invariant)chain(Ii).Ii is a type ? transmembrane glycoprotein,leukocyte differentiation antigen number CD74.As an important chaperone protein of MHC ? molecules,Ii assists in the correct folding,assembly,structural maintenance and presentation of exogenous antigenic peptides of MHC ? molecules.In the MHC and ? molecules of alpha and beta chain formation process of nine dimers in the region of CLIP(the class-? associated Ii chain peptide)occupy the MHC class ? molecules of the antigen binding groove,prevent endogenous antigen peptide and MHC class ? molecules.The interaction between Ii and MHC molecules by eukaryotic and prokaryotic expression system research.In eukaryotic expression,the expression of Ii and MHC molecules was observed by CO transfection with confocal laser scanning confocal microscopy and immunoprecipitation.Due to the need to detect the molecular affinity between these two molecules,the existing eukaryotic expression products of animals are difficult to meet this kind of experiment.In order to study the interaction between the two molecules,this study explored the expression of target proteins by prokaryotic expression system.First of all,according to the GenBank Mhc ? duck alpha(accession number:AB115244),Mhc ?(DQ490138)and beta Ii(HQ909102)gene sequence,using the designed primers,and extracted RNA from duck spleen,the reverse transcription cDNA its amplified full-length duck Mhc ? alpha,? beta and Mhc Ii gene the coding region,the cloned gene fragment were inserted into the prokaryotic expression vector pET-32a and pGEX-4T-1,and the eukaryotic expression vector pEGFP-Nl and pmCherry-N1,were used for transformation of Escherichia coli Rosetta(DE3)and transfection of eukaryotic expression eukaryotic protein.By PCR,enzyme digestion and sequencing results showed that the successfully constructed 6 recombinant plasmids.Secondly,the prokaryotic expression products respectively after repeated freeze-thaw and sonication treatment,His/D-MHC ? alpha and His/D-MHC beta ? by nickel ion affinity chromatography purified GST/D-Ii protein by GST affinnity chromatography(Glutathione Sepharose 4B)for protein purification.In SDS-PAGE electrophoresis,His/D-MHCI protein molecular weight of 57.7 kD,His/D-MHC ?protein molecular weight of 46.7 kD,GST/D-Ii 50.5 kD protein,GFP-D-MHCI protein molecular weight of 54.84 kD,GFP-D-MHC ? beta 56.04 kD protein,pmCherry-D-Ii protein molecular weight is 51.11kD,its size is consistent with theoretical expectations.Then,the corresponding proteins expressed by eukaryotic expression were detected by immunoblotting.The results showed that the antibodies induced by these prokaryotic expression products could recognize and express eukaryotic protein.Thirdly,the recombinant plasmid pEGFP-N1-D-Mhc ? alpha,pEGFP-N1-Mhc ?beta or pmCherry-Nl-D-Ii were transfected into 293T cells respectively,and their expression in eukaryotic cells and the localization of their expressed products in cells were observed.The results show that both of them can be expressed in cells,and distributed in membrane systems.Finally,with the combination function for the detection of prokaryotic expression of Mhc ? and Ii Mhc alpha,? beta,PET-32a-D-Mhc alpha and PET-32a-D-Mhc beta ? ? and pGEX-4T-1-D-Ii were transformed into Escherichia coli,the induced expression and purification of refolded Mhc ? alpha and Mhc beta ? were mixed with Ii incubation,extraction chromatography the complexes of nickel ion affinity.The results show that the co expression product by affinity column purification,in untreated SDS PAGE,compound Mhc ?/Ii and Mhc ? alpha beta/Ii,treated with SDS and subjected to electrophoresis,found in SDS-PAGE in these complexes were dissociated into 2 corresponding monomer bands.These results show that the duck Mhc ? alpha,? beta Mhc and Ii gene in prokaryotic and eukaryotic expression of the product can maintain its antigenicity,and prokaryotic coexpression of Mhc ? alpha,Mhc beta and Ii ? protein after refolding in Pull-down showed that they combine with each other.The results of this study provide a method for further study of Mhc ?,Mhc ? and Ii alpha beta molecular relationship.