title: Synthesis of chicken major histocompatibility complex class II oligomers using a baculovirus expression system.
abstract: Chicken major histocompatibility complex (MHC) B21 and B19 haplotypes are associated with resistance and susceptibility to Marek's disease (MD), respectively. T-cell-mediated immune response is crucial in coordinating protection against Marek's disease virus (MDV) infection, but it has been difficult to identify and characterize antigen-specific T-cells. MHC class II tetramers and oligomers have been widely used for characterization of antigen-specific T-cells in the context of infectious and autoimmune diseases. Thus, the objective of this study was to synthesize chicken MHC class II oligomers of B21 and B19 haplotypes for the future identification of antigen-specific T-cells. To achieve this objective, full-length coding sequences of chicken MHC class II B21 and B19 molecules were amplified and the molecules were expressed as fusion proteins, carrying Fos and Jun leucine zipper (LZ), histidine-tag and biotin ligase recognition site sequences, using a baculovirus expression system. Recombinant MHC-II were loaded with self-peptides, which stabilized the heterodimer in SDS-PAGE and allowed the detection of these molecules in Western blots with a conformation-specific anti-chicken MHC class II antibody. Biotinylated MHC molecules were conjugated to streptavidin to form oligomers, which were resolved under the transmission electron microscope through immuno-gold labelling, thus confirming success of oligomerization. In conclusion, chicken MHC class II oligomers may be used in the future to study the antigen-specific CD4+ T-cell compartment.
authors.affiliation: Department of Pathobiology, University of Guelph, Guelph, Ont., Canada N1G 2W1.
authors.forename: Patrycja K
authors.initials: PK
authors.lastname: Niemiec
authors.forename: L R
authors.initials: LR
authors.lastname: Read
authors.forename: Shayan
authors.initials: S
authors.lastname: Sharif
mesh_terms: D000818:Animals; D016367:Baculoviridae; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002645:Chickens / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006239:Haplotypes; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016350:Leucine Zippers / Q000235:genetics; D008380:Marek Disease / Q000235:genetics / Q000276:immunology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D018411:Spodoptera
chemical_list: D000949:Histocompatibility Antigens Class II; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins