Study On The Binding Of Chicken Ii Fragments To MHC Molecules And Assisting Antigen Transport

The major histocompatibility complex(MHC)has antigen processing and presentation in immune response for animals.The MHC class Ⅱ chaperone invariant chain(Ii)acts on MHC class Ⅱ molecules in the maturity,assembly and proper folding.The Ii structure of different species is similar,and the homology is above 86%.Ii consists of four domains:the transmembrane region(Cyt),the cytoplasmic region(Tra),the CLIP region(class Ⅱ-associated invariant chain peptide,CLIP)and the trimer region(Trim).It is found that the vaccines based on Ii as carrier can stimulate the animals to produce higher antibody levels.This work focus on the binding characteristics of chicken Ii to MHC classⅡ molecules from other different species,the role in assisting intracellular antigen transport and its trigger cellular pathway.First,the characteristics of cross binding of chicken Ii fragments to MHC molecules were studied.The primers were designed based on the gene sequences of Ii,Mhc la,and Mhc Ⅱβ of mouse(H2),chicken(B)and grass carp,which were registered in GenBank.The full-length gene sequences of them were amplified by PCR and cloned into eukaryotic expression vector(pEGFP/pmCherry-C1/N1)respectively to constructe recombinant eukaryotic plasmids.Then they were transfected into 293T cells respectively,and the intracellular colocalization of Ii and MHC molecules was observed by laser confocal microscopy.The results showed that mouse,chicken,grass carp Iis could colocalized with MHC la,MHC Ⅱβ molecules as a cross-species form in the transfected cells.Moreover,mouse Ii(mIi),chicken Ii(cIi)and grass carp Ii(gIi)were constructed into the prokaryotic expression vector pGEX-4T-1,respectively,and then the reconstruted expression plasmids(pGEX-4T-1-mIi,pGEX-4T-1-cIi and pGEX-4T-1-gIi were constructed.According to the structure of Mhc Ⅱβ gene of different species,the gens of antigen peptide binding domain(β1)were constructed into the prokaryotic expression vector pET-32a respectively to construct the eukaryotic recombinant plasmids(pET-32a-mβ1,pET-32a-cβl and pET-32a-gβ1).The plasmids were transformed into E.coli Rosetta(DE3),and then the recombinant bacteria were induced to express target proteins respectively.The expressed proteins were purified by Ni-graphy column,and the characteristics of binding Ii to β1 between various species were detected by Pull-down technique.The mice and chickens of different species were found.Grass carp Ii can cross-mature MHC Ⅱβ 1 in vitro.Secondly,the study was to detect the transport of antigenic peptides in the cytoplasmic/transmembrane region(Ii(Cyt/Tra))of chicken Ii.In this study,four gene target fragments(Ii,Ii(Cyt/Tra),F2(Newcastle disease virus F protein fragment)and Ii(Cyt/Tra)/F2)were constructed and inserted into prokaryotic and eukaryotic expression vector PET32a and pmCherry-C1/N1 respectively.Then eight recombinant prokaryotic and eukaryotic plasmids were constructed.The recombinant prokaryotic plasmids were transformed into E.coli Rosetta(DE3),and the binding of target protein(Ii)to MHC Ⅱβmolecules was detected by pull-down method.The four recombinant eukaryotic were transfected into eukaryotic cells 293T,and then the colocalization of these Ii molecules with MHC Ⅱβ in the endosomes of the cells was determined by laser confocal microscopy.The results showed that the constructed recombinant plasmids could express the corresponding target proteins in prokaryotic and eukaryotic cells;Ii,Ii(Cyt/Tra)and Ii(Cyt/Tra)/F2 could not only bind to MHC Ⅱβ but also enter the endosomes of cells,but F2 could neither interact with MHC Ⅱβ nor enter endosomes.It is indicated that the active fragment of Ii,Cyt/Tra,not only had the function of binding to MHC Ⅱβ,but also could carry the antigen peptide to bind to MHC Ⅱβ and transfer F2 into the endosomes and also to enter the antigen presentation pathway.Finally,the effects of signaling pathways that cause immune responses in the Ii functional region were explored.The eukaryotic plasmid pmCherry-Cl-cIi(Cyt/Tra)was transfected into chicken macrophage HD-11.After 24 hours,the RNA was extracted and reverse transcription carried out.Real-time quantitative PCR was used to detect the relevant membrane receptor(TLR4,NLRC5 and NLRP3),partial antigen presenting molecules(Ii and MHC Ⅱβ),signaling pathway molecules(STAT3 and NF-κB),and cytokines(IL-6,TNF-a,IL-1β).The results showed that the Ii(Cyt/Tra)as Ii functional fragment could up-regulate the transcription level of NF-κB gene,at same time up-regulate the transcription levels of Ii,MHC Ⅱβ,NLRP3,IL-6,TNF-a and IL-1β genes,but down-regulate of the expression of STAT3.Because NF-κB is an important regulative factor in immune response,it is speculated that the Ii function domain might promote the expression of antibody-presenting molecules and their cytokines via regulating the NF-κB signaling pathway and thus increase cellular immunity.All these results provide a theoretical basis for Ii and its functional fragments as immunological vectors,and for further exploring Ii as immune vector in vaccine in enhancement of immune effect.