Characters Of The Location In Cells And Binding Activity Of Chicken Ii And B-L Gene Expressed Products

Major histocompatibility complex(MHC)is some protein molecules encoded by a closely linked gene group located in vertebrate genomes,which appear high degree of polymorpHism.MHC molecules distribute on various cell surface,present antigen peptides and regulate immune response.In the different animals it and its encoding products are given special names.In MHC molecular family class II is consisted of two chains,? and ?,as a heterodimers.In endoplasmic reticulum it binds invariant chain(Ii)to form nonamer.Ii is a type II transmembrane glycoprotein,its leukocyte differentiation antigen is CD74.Ii assists MHC II molecules to correct folding,combination,structure maintains,present exogenous antigen peptide.The CLIP region(the class-II associated Ii chain peptide)in Ii occupies the antigen binding groove of MHC II molecules during aggregation ?,? chains to form polymer,which prevents endogenous peptide from associating with the MHC II class of molecules.The research of an interaction between Ii with MHC molecules is via in eukaryotic and prokaryotic expression system.In the eukaryotic system the both genes are co-transfected into cells,and expression protein and their interaction are observed by confocal laser and co-immunoprecipitation.In the study of their interaction there is an analysis of the affinity between both molecules,but the eukaryotic expression system can not need to a large number of expressed products.Therefor in this study the prokaryotic expression system was used to detect the interaction between the both molecules.First,chicken Ii(login ID: AY597053),B-LA(login ID: AY357254)and B-LB(login ID: S66480)gene sequences in GenBank was achieved.With a series of self-designed primers,whole genes of Ii,B-LA and B-L were cloned from the cDNAs saved in our laboratory respectively.These genes were further inserted into prokaryotic expression carrier pGEX-4T-1 and pET-28 a,and eukaryotic expression vector pEGFP-C1 and pEGFP-N1 respectively,which were used to transfect E.coli,Rosetta(DE3),or eukaryotic cells(293T).The result of identification by PCR,ouble-enzyme cleavage and sequencing showed that six recombinant plasmids were successfully formed.Secondly,prokaryotic expression products were repeatedly frozen and thawed,and splitted with ultrasonic treatment respectively.Then the samples were purified with Glutathione Sepharose 4B(GST-Ii fusion protein)or with Ni Sepharose TM 6 Fast Flow(His/B-LA or His/B-LB).The results of SDS-PAGE showed that molecular weights of purified proteins were 50.8 kD(GST-Ii),34.1 kD(His/B-LA),35.3 kD(His/B-LB),54.17 kD(GFP-Ii),54.84 kD(GFP-B-LA)or 56.04 kD(GFP-B-LB)respectively,which was consistent with the expected value.These purified prokaryotic expression proteins were then renatured and immunized mice respectively.The specific antiserums were achieved and used as antibody to detect eukaryotic expressed proteins in a western blot..The results indicated that the antibodies induced by prokaryotic expression proteins could recognize and bind these corresponding products in eukaryotic expression.Thirdly,the recombinant plasmids pEGFP-C1-C-Ii,pEGFP-N1-B-LA or pEGFP-N1-B-LB was transfected into 293 T cells to observe expression of object genes and the localization of their products in cells.The results showed that Ii,B-LA and B-LB could well express in cells and localize in membranae serosa.Finally,to detect function of chicken Ii,B-LA and B-LB in association the pGEX-4T-1-C-Ii and pET-28a-B-LA or pET-28a-B-LB were co-transfected into E.coli,induced to express and renatured,then the products were purified with Glutathione Sepharose 4B column to obtain the complex.The results showed that the samples treated without SDS appeared complexes Ii/B-LA and Ii/B-LB in PAGE,while the samples treated with SDS were dissociated into two monomers and as two single bands in SDS-PAGE.All of these indicated that the prokaryotic or eukaryotic expressed products of chicken Ii and B-L genes could retain their antigenicity,while in Pull-down method the prokaryotic coexpressed chicken Ii and B-L molecules could bind to form complex after their renaturation.The results in this work provide a useful method to study the relation between Ii and MHC molecules.